JP6800208B2 - PD marker for hepatocyte growth factor (HGF) - Google Patents
PD marker for hepatocyte growth factor (HGF) Download PDFInfo
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- JP6800208B2 JP6800208B2 JP2018505994A JP2018505994A JP6800208B2 JP 6800208 B2 JP6800208 B2 JP 6800208B2 JP 2018505994 A JP2018505994 A JP 2018505994A JP 2018505994 A JP2018505994 A JP 2018505994A JP 6800208 B2 JP6800208 B2 JP 6800208B2
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- apoa4
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Description
本発明は、肝細胞増殖因子（以下、ＨＧＦとも称する）のＰＤマーカー、およびその利用に関する。 The present invention relates to a PD marker for hepatocyte growth factor (hereinafter, also referred to as HGF) and its use.
ＨＧＦは、ヒト劇症肝炎患者の血漿から精製された肝実質細胞増殖活性を有する因子であり（特許文献１、および非特許文献１）、抗腫瘍効果、細胞性免疫の増強、創傷治療効果、組織の再生促進効果など、様々な薬理学的効果があることが報告されている（例えば、特許文献２）。 HGF is a factor having hepatic parenchymal cell proliferative activity purified from plasma of human fulminant hepatitis patients (Patent Document 1 and Non-Patent Document 1), and has antitumor effect, enhancement of cell-mediated immunity, wound healing effect, It has been reported that it has various pharmacological effects such as a tissue regeneration promoting effect (for example, Patent Document 2).
薬剤開発の確度を高めるために、薬力学的作用を反映するバイオマーカー（ＰＤマーカー）の開発は重要であり、ＨＧＦについても、これまでに肝臓障害を有する患者に対するＨＧＦの薬効をモニタリングするための指標としてα−フェトプロテインおよび可溶性Ｆａｓが提案されている（特許文献３）。 In order to increase the accuracy of drug development, it is important to develop biomarkers (PD markers) that reflect pharmacodynamic effects, and for HGF as well, to monitor the efficacy of HGF in patients with liver damage so far. Alpha-fetoprotein and soluble Fas have been proposed as indicators (Patent Document 3).
しかし、これらのマーカーは、いずれも肝臓疾患の病態改善が反映される血中マーカーであることから、投与されたＨＧＦが薬力学的作用を示していることを示すものではない。 However, since all of these markers are blood markers that reflect the improvement of the pathological condition of liver disease, they do not indicate that the administered HGF has a pharmacodynamic effect.
本発明は、より広範な対象においてＨＧＦの投与によって変動が認められるＰＤマーカーを利用した、ＨＧＦの薬理学的作用を評価する方法を提供することを目的とする。 An object of the present invention is to provide a method for evaluating the pharmacological action of HGF using a PD marker whose fluctuation is observed by administration of HGF in a wider range of subjects.
本発明者らは、上記課題を解決するために鋭意研究を重ねる過程で、ＨＧＦ投与によって発現が変動するマーカーの１つとしてアポリポタンパク質Ａ−ＩＶ（ＡｐｏＡ４）を見出した。本発明者らは、ＡｐｏＡ４についてさらなる研究を進めたところ、ＡｐｏＡ４遺伝子が、ＨＧＦとｃ−Ｍｅｔとの相互作用により発現が誘導されること、ＡｐｏＡ４遺伝子発現の誘導が血液中のＡｐｏＡ４タンパク質で検出できること、および正常なヒト肝細胞においてもＨＧＦの暴露によってＡｐｏＡ４遺伝子発現の増加が認められるマーカーであることを見出した。 The present inventors have found apolipoprotein A-IV (ApoA4) as one of the markers whose expression is changed by HGF administration in the process of conducting intensive studies to solve the above problems. As a result of further research on ApoA4, the present inventors have found that the expression of the ApoA4 gene is induced by the interaction between HGF and c-Met, and that the induction of the expression of the ApoA4 gene can be detected by the ApoA4 protein in blood. , And also in normal human hepatocytes, it was found that it is a marker in which an increase in ApoA4 gene expression is observed by exposure to HGF.
本発明は上記知見に基づくものであり、以下の態様を包含する。
［１］ 肝細胞増殖因子（ＨＧＦ）の薬力学的作用を評価する方法であって、
ＨＧＦが投与された被験体から採取した生体試料中のＡｐｏＡ４のレベルを測定するステップを含む、
ことを特徴とする、
評価方法。The present invention is based on the above findings and includes the following aspects.
[1] A method for evaluating the pharmacodynamic effect of hepatocyte growth factor (HGF).
Including the step of measuring the level of ApoA4 in a biological sample taken from a subject to which HGF was administered.
Characterized by
Evaluation method.
［２］ ［１］に記載の評価方法であって、
ＨＧＦが投与された被験体から採取した生体試料中のＡｐｏＡ４のレベルを、ＨＧＦが投与されていない、または、その投与の影響が消失した被験体から採取した前記生体試料中のＡｐｏＡ４のレベルと比較するステップをさらに含む、
ことを特徴とする、
評価方法。[2] The evaluation method according to [1].
Compare the level of ApoA4 in a biological sample taken from a subject to whom HGF was administered to the level of ApoA4 in the biological sample taken from a subject who was not given HGF or had no effect of its administration. Including more steps to do,
Characterized by
Evaluation method.
［３］ ［２］に記載の評価方法であって、
ＨＧＦが投与されていない、または、その投与の影響が消失した被験体のＡｐｏＡ４のレベルと比較して、ＨＧＦが投与された被験体のＡｐｏＡ４のレベルが上昇している場合に、当該投与されたＨＧＦが薬力学的作用をもたらす量で投与されていると判定するステップをさらに含む、
ことを特徴とする、
評価方法。[3] The evaluation method according to [2].
If the level of ApoA4 in the subject to whom HGF was administered was elevated compared to the level of ApoA4 in the subject who was not administered HGF or the effects of the administration disappeared, the dose was administered. Further including the step of determining that HGF is administered in an amount that produces a pharmacodynamic effect,
Characterized by
Evaluation method.
［４］ ［１］〜［３］のいずれかに記載の評価方法であって、
前記「ＡｐｏＡ４のレベル」は、ＡｐｏＡ４タンパク質の発現レベルである、
ことを特徴とする、
評価方法。[4] The evaluation method according to any one of [1] to [3].
The "ApoA4 level" is the expression level of the ApoA4 protein.
Characterized by
Evaluation method.
［５］ ［４］に記載の評価方法であって、
生体由来試料中のＡｐｏＡ４タンパク質の発現レベルが、イムノアッセイによって測定される、
ことを特徴とする、
評価方法。[5] The evaluation method according to [4].
The expression level of ApoA4 protein in a biological sample is measured by an immunoassay.
Characterized by
Evaluation method.
［６］ ［１］〜［５］のいずれかに記載の評価方法であって、
前記生体由来試料は、血清または血漿に由来する試料である、
ことを特徴とする、
評価方法。[6] The evaluation method according to any one of [1] to [5].
The biological sample is a sample derived from serum or plasma.
Characterized by
Evaluation method.
［７］ ［１］〜［３］のいずれかに記載の評価方法であって、
前記「ＡｐｏＡ４のレベル」は、ＡｐｏＡ４遺伝子の発現レベルである、
ことを特徴とする、
評価方法。[7] The evaluation method according to any one of [1] to [3].
The "level of ApoA4" is the expression level of the ApoA4 gene.
Characterized by
Evaluation method.
［８］ ［７］に記載の評価方法であって、
生体由来試料中のＡｐｏＡ４遺伝子の発現レベルが、リアルタイムＰＣＲによって測定される、
ことを特徴とする、
評価方法。[8] The evaluation method according to [7].
The expression level of the ApoA4 gene in a biological sample is measured by real-time PCR.
Characterized by
Evaluation method.
［９］ ［１］〜［３］、[７]または[８]のいずれかに記載の評価方法であって、
前記生体試料は、肝細胞または肝組織である、
ことを特徴とする、
評価方法。[9] The evaluation method according to any one of [1] to [3], [7] or [8].
The biological sample is hepatocytes or liver tissue.
Characterized by
Evaluation method.
［１０］ ［１］〜［９］のいずれかに記載の評価方法であって、
前記被験体は、健常人である、
ことを特徴とする、
評価方法。[10] The evaluation method according to any one of [1] to [9].
The subject is a healthy person,
Characterized by
Evaluation method.
［１１］ ［１］〜［９］のいずれかに記載の評価方法であって、
前記被験体は、肝臓疾患を有する被験体である
ことを特徴とする、
評価方法。[11] The evaluation method according to any one of [1] to [9].
The subject is characterized by having a liver disease.
Evaluation method.
［１２］ ＨＧＦの薬力学的作用を評価するためのキットであって、
抗ＡｐｏＡ４抗体を含む、
ことを特徴とする、
キット。[12] A kit for evaluating the pharmacodynamic effect of HGF.
Contains anti-ApoA4 antibody,
Characterized by
kit.
以上述べた本発明の一または複数の特徴を任意に組み合わせた発明も、本発明の範囲に含まれることを、当業者であれば理解するであろう。 Those skilled in the art will understand that an invention in which one or more of the features of the present invention described above are arbitrarily combined is also included in the scope of the present invention.
本発明によれば、より広範な対象において、ＨＧＦの投与によって変動が認められるＰＤマーカーとしてＡｐｏＡ４を利用した、ＨＧＦの薬力学的作用を評価する方法が提供される。 According to the present invention, there is provided a method for evaluating the pharmacodynamic effect of HGF using ApoA4 as a PD marker whose fluctuation is observed by administration of HGF in a wider range of subjects.
以下、本発明の実施の形態を具体的に説明する。 Hereinafter, embodiments of the present invention will be specifically described.
本発明は、ＨＧＦの薬力学的作用を評価する方法に関する。本発明の評価方法は、対象におけるＡｐｏＡ４のレベルが、ＨＧＦ投与によって変動することを新たに見出したことを基礎とする。したがって、本発明の評価方法は、ＨＧＦが投与された被験体から採取した生体試料中のＡｐｏＡ４のレベルを測定するステップを含むことを特徴とする。 The present invention relates to a method for assessing the pharmacodynamic effects of HGF. The evaluation method of the present invention is based on the newly found that the level of ApoA4 in a subject is changed by HGF administration. Therefore, the evaluation method of the present invention is characterized by including a step of measuring the level of ApoA4 in a biological sample collected from a subject to which HGF has been administered.
本発明に使用されるＨＧＦは、市販のものであってもよいし、組み換え技術によって製造されたものであってもよい。組み換え技術によるＨＧＦの製造は、例えば、ＨＧＦをコードする遺伝子をクローニングし、当該遺伝子を含むベクターを作製し、これを宿主細胞に導入して形質転換させることによって、ＨＧＦを発現する細胞を取得し、これを細胞培養することによって製造することができる。このプロセスに使用される細胞、ベクターの種類、細胞の種類、培養条件等は、当業者の技術的範囲内であり、適宜適切な条件を設定することができる。 The HGF used in the present invention may be commercially available or may be produced by a recombinant technique. In the production of HGF by recombinant technology, for example, a gene encoding HGF is cloned, a vector containing the gene is prepared, and the vector is introduced into a host cell for transformation to obtain a cell expressing HGF. , This can be produced by cell culture. The cells, vector type, cell type, culture conditions, etc. used in this process are within the technical scope of those skilled in the art, and appropriate conditions can be set as appropriate.
本発明において、評価の対象となる被験体は、特に制限されないが、例えば、ＨＧＦ投与の対象となる、肝臓疾患を発症している被験体、肝臓疾患を発症するおそれがある被験体、および健常者が挙げられる。本発明において、肝臓疾患は、任意の肝臓疾患であり得るが、好ましくは、重症肝障害である。被験体は、典型的にはヒトを指すが、ＡｐｏＡ４の発現が認められる動物であれば、例えば他の霊長類、げっ歯類などであってもよい。 In the present invention, the subject to be evaluated is not particularly limited, but for example, a subject who develops liver disease, a subject who may develop liver disease, and a healthy subject who are subject to HGF administration. Can be mentioned. In the present invention, the liver disease can be any liver disease, but is preferably severe liver damage. The subject typically refers to a human, but may be, for example, another primate, rodent, or the like, as long as it is an animal in which expression of ApoA4 is observed.
ＨＧＦの投与経路は、特に制限されないが、ＨＧＦがタンパク質製剤であることに鑑み、典型的には、静脈内投与である。 The route of administration of HGF is not particularly limited, but in view of the fact that HGF is a protein preparation, it is typically administered intravenously.
本発明において、前記「被験体から採取した生体試料」は、被験体から採取可能なものである限り特に限定されず、例えば組織（動物を構成する基本的な単位である細胞が特定の機能を発揮するように分化し集団を形成したもので、細胞と細胞間質によって構成され、全体としてある機能を果たすもの）、器官（組織が規則正しく組み合わされて作り上げられたもの）または体液とすることができる。組織または器官としては、非限定的に、卵巣、子宮、***、甲状腺、脳、食道、舌、肺、膵臓、胃、小腸、十二指腸、大腸、膀胱、腎臓、肝臓、前立腺、胆嚢、咽頭、筋肉、骨および皮膚などが挙げられるがこれらに限定されない。体液としては、非限定的に、血清、血漿または全血などの血液、リンパ液、組織液、髄液、体腔液、消化液、鼻水、涙液、汗および尿などが挙げられるがこれらに限定されない。取得および処理の簡便さから、前記生体試料として血清または血漿を用いることが好ましい。また、前記体液は、対象から採取した体液そのものであっても、採取した体液に通常行われる希釈、濃縮等の処理を行ったものであってもよい。なお、本発明に用いる被験体に由来する生体試料の採取や調製を行なう者は、本発明の工程を行う者と同一人物でもよく、別人物であってもよい。また、本発明に用いる被験体に由来する生体試料は、本発明の実施時に採取または調製されたものでもよく、予め採取または調製され保存されたものであってもよい。 In the present invention, the "biological sample collected from a subject" is not particularly limited as long as it can be collected from a subject, and for example, a tissue (a cell which is a basic unit constituting an animal has a specific function). It is differentiated to exert and form a group, which is composed of cells and cell stromal cells and fulfills a certain function as a whole), organs (made by regular combination of tissues) or body fluids. it can. Tissues or organs include, but are not limited to, ovary, uterus, breast, thyroid, brain, esophagus, tongue, lung, pancreas, stomach, small intestine, duodenum, large intestine, bladder, kidney, liver, prostate, gallbladder, pharynx, muscle. , Bone and skin, but not limited to these. Body fluids include, but are not limited to, blood such as serum, plasma or whole blood, lymph, tissue fluid, cerebrospinal fluid, coelomic fluid, digestive fluid, runny nose, tears, sweat and urine. From the viewpoint of ease of acquisition and processing, it is preferable to use serum or plasma as the biological sample. In addition, the body fluid may be the body fluid itself collected from the subject, or the collected body fluid may be subjected to treatments such as dilution and concentration which are usually performed. The person who collects and prepares the biological sample derived from the subject used in the present invention may be the same person as the person who performs the process of the present invention, or may be a different person. In addition, the biological sample derived from the subject used in the present invention may be one collected or prepared at the time of carrying out the present invention, or may be previously collected or prepared and stored.
本発明は、一実施形態において、ＨＧＦが投与された被験体から採取した生体試料中のＡｐｏＡ４のレベルを、ＨＧＦが投与されていない、または、その投与の影響が消失した被験体から採取した前記生体試料中のＡｐｏＡ４のレベルと比較するステップをさらに含む。「その投与の影響が消失した被験体」とは、ＨＧＦ投与歴はあるが、ＨＧＦによる薬力学的作用が消失するのに十分な時間が経過している被験体をいう。 In one embodiment, the present invention collects the level of ApoA4 in a biological sample collected from a subject to which HGF has been administered from a subject to which HGF has not been administered or the effects of the administration have disappeared. It further includes a step of comparing the level of ApoA4 in the biological sample. "Subject whose effect of administration has disappeared" means a subject who has a history of HGF administration but has passed a sufficient time for the pharmacodynamic effect of HGF to disappear.
本発明は、さらに好ましい実施形態において、ＨＧＦが投与されていない、または、その投与の影響が消失した被験体のＡｐｏＡ４のレベルと比較して、ＨＧＦが投与された被験体のＡｐｏＡ４のレベルが上昇している場合に、当該投与されたＨＧＦが薬理学的作用をもたらす量で投与されていると判定するステップをさらに含む。 In a more preferred embodiment, the present invention increases the level of ApoA4 in a subject to whom HGF has been administered as compared to the level of ApoA4 in a subject who has not been administered HGF or whose effects of administration have disappeared. If so, it further comprises the step of determining that the administered HGF is administered in an amount that produces a pharmacological effect.
本発明において、「薬力学的作用をもたらす量で投与されている」とは、ＨＧＦ投与によって何らかの薬力学的作用が提供されていることを意味し、その作用の大小は必ずしも問題にはされない。例えば、ＨＧＦ投与後にＡｐｏＡ４レベルが大きく上昇した場合も、ＡｐｏＡ４レベルがわずかに上昇したに過ぎない場合も、いずれも「薬力学的作用をもたらす量で投与されている」に該当する。この関連で、「薬力学的作用をもたらす量で投与されている」は、「治療上有効な量で投与されている」と同義ではなく、ＡｐｏＡ４レベルの上昇度に応じて、「治療上有効な量で投与されている」と判定される場合もあれば、「治療上有効な量で投与されている」と判定されない場合もある。 In the present invention, "administered in an amount that causes a pharmacodynamic effect" means that some pharmacodynamic effect is provided by HGF administration, and the magnitude of the effect does not necessarily matter. For example, both cases where ApoA4 levels are significantly increased after HGF administration and where ApoA4 levels are only slightly increased correspond to "administered in an amount that produces a pharmacodynamic effect". In this regard, "administered in an amount that produces a pharmacodynamic effect" is not synonymous with "administered in a therapeutically effective amount" and is "therapeutically effective" depending on the degree of increase in ApoA4 level. In some cases, it is determined that the dose is being administered in an appropriate amount, and in other cases, it is not determined that the dose is administered in a therapeutically effective amount.
本発明において、ＨＧＦは、ヒト、マウス、ラット、ブタ、その他の動物に由来するＨＧＦを含み得るが、好ましくはヒト由来のＨＧＦである。 In the present invention, HGF may include HGF derived from humans, mice, rats, pigs and other animals, but is preferably human-derived HGF.
本発明において、ヒトＨＧＦ（ｈＨＧＦ）には、配列番号１に示すアミノ酸配列を有するポリペプチドまたはその変異体が含まれる。ヒトＨＧＦの変異体には、配列番号１に示すアミノ酸配列において、１個または複数個のアミノ酸の付加、欠失または置換を有するアミノ酸配列を有し、かつ、配列番号１に示すアミノ酸配列を有するポリペプチドと同等またはそれ以上のＨＧＦ活性を有するポリペプチドが含まれる。ここでいう「複数個」は、２〜１５０個、より好ましくは２〜８０個、より好ましくは２〜７０個、より好ましくは２〜６０個、より好ましくは２〜５０個、より好ましくは２〜４０個、より好ましくは２〜３０個、より好ましくは２〜２０個、より好ましくは２〜１０個、またはより好ましくは２〜５個である。前記変異体には、また、配列番号１に示すアミノ酸配列と、少なくとも８０％、より好ましくは少なくとも８５％、より好ましくは少なくとも９０％、９１％、９２％、９３％、９４％、９５％、９６％、９７％、９８％、または９９％の配列同一性を示すアミノ酸配列を有し、かつ、配列番号１に示すアミノ酸配列を有するポリペプチドと同等またはそれ以上のＨＧＦ活性を有するポリペプチドまたは配列番号１に示すアミノ酸配列をコードする遺伝子とストリンジェントな条件でハイブリダイズする遺伝子によってコードされるアミノ酸配列を有し、かつ、配列番号１に示すアミノ酸配列を有するポリペプチドと同等またはそれ以上のＨＧＦ活性を有するポリペプチドが含まれる。 In the present invention, human HGF (hHGF) includes a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof. The human HGF variant has an amino acid sequence having one or more amino acid additions, deletions or substitutions in the amino acid sequence shown in SEQ ID NO: 1, and has an amino acid sequence shown in SEQ ID NO: 1. A polypeptide having HGF activity equal to or higher than that of the polypeptide is included. The term "plurality" as used herein refers to 2 to 150 pieces, more preferably 2 to 80 pieces, more preferably 2 to 70 pieces, more preferably 2 to 60 pieces, more preferably 2 to 50 pieces, and more preferably 2 pieces. ~ 40 pieces, more preferably 2-30 pieces, more preferably 2-20 pieces, more preferably 2-10 pieces, or more preferably 2-5 pieces. The variants also include the amino acid sequence set forth in SEQ ID NO: 1, at least 80%, more preferably at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%. A polypeptide having an amino acid sequence showing 96%, 97%, 98%, or 99% sequence identity and having HGF activity equal to or higher than that of the amino acid sequence shown in SEQ ID NO: 1. It has an amino acid sequence encoded by a gene encoding the amino acid sequence shown in SEQ ID NO: 1 and a gene that hybridizes under stringent conditions, and is equal to or higher than that of a polypeptide having the amino acid sequence shown in SEQ ID NO: 1. Polypeptides with HGF activity are included.
本発明において、「ストリンジェントな条件」とは、ポストハイブリダイゼーションの洗浄において、例えば「２×ＳＳＣ、０．１％ＳＤＳ、５０℃」の条件、「２×ＳＳＣ、０．１％ＳＤＳ、４２℃」の条件、または「１×ＳＳＣ、０．１％ＳＤＳ、３７℃」の条件における洗浄でハイブリダイズしていること、よりストリンジェントな条件としては、例えば「２×ＳＳＣ、０．１％ＳＤＳ、６５℃」、「０．５×ＳＳＣ、０．１％ＳＤＳ、４２℃」、「０．２×ＳＳＣ、０．１％ＳＤＳ、６５℃」、または「０．１×ＳＳＣ、０．１％ＳＤＳ、６５℃」の条件における洗浄でハイブリダイズしていることが挙げることができる（１×ＳＳＣは１５０ ｍＭ ＮａＣｌ、１５ ｍＭ ｓｏｄｉｕｍ ｃｉｔｒａｔｅ、ｐＨ ７．０）。より詳細には、Ｒａｐｉｄ−ｈｙｂ ｂｕｆｆｅｒ（Ａｍｅｒｓｈａｍ Ｌｉｆｅ Ｓｃｉｅｎｃｅ）を用いた方法として、６８℃で３０分以上プレハイブリダイゼーションを行った後、プローブを添加して１時間以上６８℃に保ってハイブリッドを形成させ、その後、２×ＳＳＣ、０．１％ＳＤＳ中、室温で２０分の洗浄を３回、１×ＳＳＣ、０．１％ＳＤＳ中、３７℃で２０分の洗浄を３回、最後に、１×ＳＳＣ、０．１％ＳＤＳ中、５０℃で２０分の洗浄を２回行うことも考えられる。より好ましくは、プレハイブリダイゼーションおよびハイブリダイゼーションの溶液としては、例えば５×ＳＳＣ、７％（Ｗ／Ｖ）ＳＤＳ、１００μｇ／ｍｌ変性サケ***ＤＮＡ、５×デンハルト液（１×デンハルト溶液は０．２％ポリビニールピロリドン、０．２％牛血清アルブミン、及び０．２％フィコールを含む）を含む溶液を用い、プレハイブリダイゼーションを６５℃３０分から１時間、ハイブリダイゼーションを同じ温度で一晩（６〜８時間）行う。その他、例えばＥｘｐｒｅｓｓｈｙｂ Ｈｙｂｒｉｄｉｚａｔｉｏｎ Ｓｏｌｕｔｉｏｎ（ＣＬＯＮＴＥＣＨ）中、５５℃で３０分以上プレハイブリダイゼーションを行い、標識プローブを添加し、３７〜５５℃で１時間以上インキュベートし、２×ＳＳＣ、０．１％ＳＤＳ中、室温で２０分の洗浄を３回、１×ＳＳＣ、０．１％ＳＤＳ中、３７℃で２０分の洗浄を１回行うこともできる。ここで、例えば、プレハイブリダイゼーション、ハイブリダイゼーションや２度目の洗浄の際の温度を上げることにより、よりストリンジェントな条件とすることができる。例えば、プレハイブリダイゼーション及びハイブリダイゼーションの温度を６０℃、さらにストリンジェントな条件としては６５℃または６８℃とすることができる。当業者であれば、このようなバッファーの塩濃度、温度等の条件に加えて、その他のプローブ濃度、プローブの長さ、反応時間等の諸条件を加味し、本発明の遺伝子のアイソフォーム、アレリック変異体、及び対応する他種生物由来の遺伝子を得るための条件を設定することができる。ハイブリダイゼーション法の詳細な手順については、『Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ， Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ ２ｎｄ ｅｄ．』（Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｐｒｅｓｓ （１９８９）；特にＳｅｃｔｉｏｎ９．４７−９．５８）、『Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ』（Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ （１９８７−１９９７）；特にＳｅｃｔｉｏｎ６．３−６．４）、『ＤＮＡ Ｃｌｏｎｉｎｇ １： Ｃｏｒｅ Ｔｅｃｈｎｉｑｕｅｓ， Ａ Ｐｒａｃｔｉｃａｌ Ａｐｐｒｏａｃｈ ２ｎｄ ｅｄ．』（Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ （１９９５）；条件については特にＳｅｃｔｉｏｎ２．１０）等を参照することができる。 In the present invention, the "stringent condition" means, for example, the condition of "2 × SSC, 0.1% SDS, 50 ° C.", "2 × SSC, 0.1% SDS, 42" in the washing of post-hybridization. Hybridization by washing under the condition of "° C." or "1 x SSC, 0.1% SDS, 37 ° C.", and more stringent conditions include, for example, "2 x SSC, 0.1%". SDS, 65 ° C, "0.5 x SSC, 0.1% SDS, 42 ° C," "0.2 x SSC, 0.1% SDS, 65 ° C," or "0.1 x SSC, 0. It can be mentioned that hybridization is performed by washing under the condition of "1% SDS, 65 ° C." (1 × SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0). More specifically, as a method using Rapid-hyb buffer (Amersham Life Science), prehybridization was performed at 68 ° C. for 30 minutes or longer, and then a probe was added and kept at 68 ° C. for 1 hour or longer to form a hybrid. Then, wash for 20 minutes at room temperature 3 times in 2 × SSC, 0.1% SDS, and wash for 20 minutes at 37 ° C. 3 times in 1 × SSC, 0.1% SDS, and finally, It is also conceivable to perform two 20-minute washes at 50 ° C. in 1 × SSC, 0.1% SDS. More preferably, the prehybridization and hybridization solutions include, for example, 5 × SSC, 7% (W / V) SDS, 100 μg / ml denatured salmon sperm DNA, 5 × Denhardt solution (1 × Denhardt solution is 0.2). Using a solution containing% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% ficol), prehybridize at 65 ° C. for 30 minutes to 1 hour and hybridize overnight at the same temperature (6 to 6 to 6). 8 hours). In addition, for example, in Expressionhyb Hybridization Solution (CLONTECH), prehybridization was performed at 55 ° C. for 30 minutes or longer, a labeled probe was added, and the mixture was incubated at 37 to 55 ° C. for 1 hour or longer in 2 × SSC, 0.1% SDS. It is also possible to perform three 20-minute washes at room temperature and one 20-minute wash at 37 ° C. in 1 × SSC, 0.1% SDS. Here, for example, by raising the temperature at the time of pre-hybridization, hybridization or the second washing, more stringent conditions can be obtained. For example, the temperature of prehybridization and hybridization can be 60 ° C., and further stringent conditions can be 65 ° C. or 68 ° C. Those skilled in the art will consider the conditions such as the salt concentration and temperature of the buffer, as well as other conditions such as the probe concentration, the probe length, and the reaction time, and the isoform of the gene of the present invention. Conditions for obtaining genes derived from allelic mutants and corresponding other species can be set. For the detailed procedure of the hybridization method, refer to "Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989); especially Section 9.47-9.58), "Curent Protocols in Molecular Biology" (John Wiley & Sons (1987-1997); especially Section 6.3-6. Cloning 1: Core Techniques, A Practical Approach 2nd ed. (Oxford University (1995); in particular, Section 2.10 for conditions) and the like can be referred to.
「ＡｐｏＡ４遺伝子」は、アポリポタンパク質Ａ−ＩＶをコードする遺伝子であり、ヒトのほか、マウス、ラット等のげっ歯類などでも発現することが知られている。ヒトＡｐｏＡ４の遺伝子配列およびそれに対応するアミノ酸配列は、それぞれ、ＧｅｎＢａｎｋ Ａｃｃｅｓｓｉｏｎ Ｎｏ．ＮＭ＿０００４８２、およびＧｅｎＢａｎｋ Ａｃｃｅｓｓｉｏｎ Ｎｏ．ＮＰ＿０００４７３で登録されている。 The "ApoA4 gene" is a gene encoding the apolipoprotein A-IV, and is known to be expressed not only in humans but also in rodents such as mice and rats. The gene sequence of human ApoA4 and the corresponding amino acid sequence are described in GenBank Accession No. NM_000482 and GenBank Accession No. It is registered at NP_000473.
本発明において、ヒトＡｐｏＡ４タンパク質は、その変異体を含んでもよい。ヒトＡｐｏＡ４タンパク質の変異体には、配列番号３に示すアミノ酸配列において、１個または複数個のアミノ酸の付加、欠失または置換を有するアミノ酸配列を有し、かつ、配列番号３に示すアミノ酸配列を有するヒトＡｐｏＡ４タンパク質と同等の機能特性を有するポリペプチドをコードするものが含まれる。ここでいう「複数個」は、２〜８０個、より好ましくは２〜６０個、より好ましくは２〜４０個、より好ましくは２〜３５個、より好ましくは２〜３０個、より好ましくは２〜２５個、より好ましくは２〜２０個、より好ましくは２〜１５個、より好ましくは２〜１０個、またはより好ましくは２〜５個である。前記変異体には、また、配列番号３に示すアミノ酸配列と、少なくとも８０％、より好ましくは少なくとも８５％、より好ましくは少なくとも９０％、９１％、９２％、９３％、９４％、９５％、９６％、９７％、９８％、または９９％の配列同一性を示すアミノ酸配列を有し、かつ、配列番号３に示すアミノ酸配列を有するヒトＡｐｏＡ４タンパク質と同等の機能特性を有するポリペプチドまたは配列番号３に示すアミノ酸配列をコードする遺伝子（例えば、配列番号２）とストリンジェントな条件でハイブリダイズする遺伝子によってコードされるアミノ酸配列を有し、かつ、配列番号３に示すアミノ酸配列を有するヒトＡｐｏＡ４タンパク質と同等の機能特性を有するポリペプチドが含まれる。例示的な実施形態において、ヒトＡｐｏＡ４タンパク質の変異体は、配列番号５に示すアミノ酸配列、または配列番号４に示す塩基配列によってコードされるアミノ酸配列を有している。 In the present invention, the human ApoA4 protein may contain a variant thereof. The variant of the human ApoA4 protein has an amino acid sequence having one or more amino acid additions, deletions or substitutions in the amino acid sequence shown in SEQ ID NO: 3, and has an amino acid sequence shown in SEQ ID NO: 3. It includes those encoding polypeptides having functional properties equivalent to those of the human ApoA4 protein. The term "plurality" as used herein refers to 2 to 80 pieces, more preferably 2 to 60 pieces, more preferably 2 to 40 pieces, more preferably 2 to 35 pieces, more preferably 2 to 30 pieces, and more preferably 2 pieces. ~ 25 pieces, more preferably 2-20 pieces, more preferably 2-15 pieces, more preferably 2-10 pieces, or more preferably 2-5 pieces. The variant also includes the amino acid sequence set forth in SEQ ID NO: 3, at least 80%, more preferably at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%. A polypeptide or SEQ ID NO: having an amino acid sequence showing 96%, 97%, 98%, or 99% sequence identity and having functional properties equivalent to those of a human ApoA4 protein having the amino acid sequence shown in SEQ ID NO: 3. A human ApoA4 protein having an amino acid sequence encoded by a gene encoding the amino acid sequence shown in 3 (for example, SEQ ID NO: 2) and a gene that hybridizes under stringent conditions, and having the amino acid sequence shown in SEQ ID NO: 3. Contains polypeptides having the same functional properties as. In an exemplary embodiment, the variant of the human ApoA4 protein has the amino acid sequence set forth in SEQ ID NO: 5 or the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 4.
前記生体試料中のＡｐｏＡ４レベルの測定は、ＡｐｏＡ４を定量的に検出できる方法であれば特に制限されない。したがって、ＡｐｏＡ４タンパク質の発現レベルを測定することによってもよいし、ＡｐｏＡ４遺伝子の発現レベル（すなわちＡｐｏＡ４ ｍＲＮＡ）を測定することによってもよい。ＡｐｏＡ４タンパク質は、ＨＧＦ投与による変動を血液中のタンパク質で確認することができるため、簡便さの点で、血清または血漿中のＡｐｏＡ４タンパク質の量を測定することが有利であり得る。 The measurement of the ApoA4 level in the biological sample is not particularly limited as long as it is a method capable of quantitatively detecting ApoA4. Therefore, the expression level of the ApoA4 protein may be measured, or the expression level of the ApoA4 gene (that is, ApoA4 mRNA) may be measured. Since the fluctuation of ApoA4 protein due to HGF administration can be confirmed by the protein in blood, it may be advantageous to measure the amount of ApoA4 protein in serum or plasma in terms of convenience.
ＡｐｏＡ４タンパク質の発現レベルの測定方法は、ＡｐｏＡ４タンパク質を特異的に検出し、測定できる方法であれば特に制限はなく、通常用いられる測定方法を適用することができる。そのような方法として、これに限定されるものではないが、ＡｐｏＡ４タンパク質に結合する抗体を用いる方法、イオン交換クロマトグラフィー、質量分析、ＬＣ／ＭＳ、ＬＣ／ＭＳに質量分析計を直列に結合したＬＣ／ＭＳ／ＭＳ等のＡｐｏＡ４タンパク質またはその断片を測定する方法等を挙げることができる。 The method for measuring the expression level of the ApoA4 protein is not particularly limited as long as it can specifically detect and measure the ApoA4 protein, and a commonly used measuring method can be applied. Such methods include, but are not limited to, methods using antibodies that bind to the ApoA4 protein, ion exchange chromatography, mass spectrometry, LC / MS, and LC / MS coupled with a mass spectrometer in series. Examples thereof include a method for measuring an ApoA4 protein such as LC / MS / MS or a fragment thereof.
本発明において、ＡｐｏＡ４タンパク質の検出に用いる装置には特に制限はなく、ＡｐｏＡ４タンパク質の測定の方法に応じて適宜選択することができる。具体的には例えば、ＨＰＬＣ機器、質量分析機器（マススペクトロメトリー）、液体クロマトグラフィーと質量分析機器を接続したＬＣ／ＭＳ機器、ＬＣ／ＭＳ機器に質量分析計を直列に結合したＬＣ／ＭＳ／ＭＳ機器、電気泳動機器（キャピラリー電気泳動装置等）、全自動あるいは半自動酵素免疫測定機器、セルウォッシャー、全自動あるいは半自動化学発光免疫測定機器、発光測定装置、全自動あるいは半自動電気化学発光免疫測定機器、光学測定装置、プレートリーダー、ＣＣＤカメラ、全自動あるいは半自動蛍光免疫測定機器、蛍光測定装置、全自動あるいは半自動放射免疫測定機器、液体シンチレーションカウンター、クールターカウンター、表面プラズモン測定装置、ブロッティング装置、デンシトメーター等を挙げることができる。 In the present invention, the apparatus used for detecting the ApoA4 protein is not particularly limited, and can be appropriately selected depending on the method for measuring the ApoA4 protein. Specifically, for example, HPLC equipment, mass spectrometry equipment (mass spectrometry), LC / MS equipment in which liquid chromatography and mass spectrometry equipment are connected, and LC / MS / in which a mass spectrometer is connected in series to LC / MS equipment. MS equipment, electrophoresis equipment (capillary electrophoresis equipment, etc.), fully automatic or semi-automatic enzyme immunoassay equipment, cell washer, fully automatic or semi-automatic chemical luminescence immunoassay equipment, luminescence measurement equipment, fully automatic or semi-automatic electrochemical luminescence immunoassay equipment , Optical measuring device, plate reader, CCD camera, fully automatic or semi-automatic fluorescence immunoassay device, fluorescence measuring device, fully automatic or semi-automatic radioimmunoassay measuring device, liquid scintillation counter, cooler counter, surface plasmon measuring device, blotting device, densi A meter or the like can be mentioned.
本発明の一実施形態において、ＡｐｏＡ４タンパク質の発現レベルの測定には、ＡｐｏＡ４タンパク質に結合する抗体（本明細書中、「抗ＡｐｏＡ４抗体」とも称する）を用いる方法、例えば抗ＡｐｏＡ４抗体を用いるイムノアッセイを使用することが好ましい。抗ＡｐｏＡ４抗体は、ＡｐｏＡ４を認識して結合可能な抗体であれば特に制限されない。 In one embodiment of the present invention, for measuring the expression level of ApoA4 protein, a method using an antibody that binds to ApoA4 protein (also referred to as "anti-ApoA4 antibody" in the present specification), for example, an immunoassay using an anti-ApoA4 antibody is used. It is preferable to use it. The anti-ApoA4 antibody is not particularly limited as long as it is an antibody that can recognize and bind to ApoA4.
抗ＡｐｏＡ４抗体は、モノクローナル抗体およびポリクローナル抗体のいずれも公知の方法に従って作製することができる。モノクローナル抗体は、例えば、ＡｐｏＡ４またはＡｐｏＡ４フラグメントで免疫した非ヒト哺乳動物から抗体産生細胞を単離し、これを骨髄腫細胞等と融合させてハイブリドーマを作製し、このハイブリドーマが産生した抗体を精製することによって得ることができる。また、ポリクローナル抗体は、ＡｐｏＡ４タンパク質またはＡｐｏＡ４フラグメントで免疫した動物の血清から得ることができる。ＡｐｏＡ４フラグメントはＡｐｏＡ４タンパク質の部分ペプチドであり、抗ＡｐｏＡ４フラグメント抗体は、ＡｐｏＡ４タンパク質を認識する。また、免疫原としては、例えば、ヒトやサルなどの霊長類、ラットやマウスなどのげっ歯類などのＡｐｏＡ４タンパク質またはＡｐｏＡ４フラグメントが挙げられるが、これらに限定されない。 Anti-ApoA4 antibody can be prepared according to a known method for both monoclonal antibody and polyclonal antibody. For monoclonal antibodies, for example, antibody-producing cells are isolated from non-human mammals immunized with ApoA4 or ApoA4 fragments, and these are fused with myeloma cells to prepare hybridomas, and the antibodies produced by these hybridomas are purified. Can be obtained by Also, polyclonal antibodies can be obtained from the sera of animals immunized with the ApoA4 protein or ApoA4 fragment. The ApoA4 fragment is a partial peptide of the ApoA4 protein and the anti-ApoA4 fragment antibody recognizes the ApoA4 protein. In addition, examples of immunogens include, but are not limited to, ApoA4 proteins or ApoA4 fragments of primates such as humans and monkeys, and rodents such as rats and mice.
なお、本明細書において「抗体」とは、天然に存在するか、遺伝子組換え技術によって産生される、全長の免疫グロブリン分子、または抗体フラグメントのような免疫グロブリン分子の免疫学的に活性な断片をいう。これらの抗体は、慣用技術を用いて作製することができ、ポリクローナル抗体であっても、モノクローナル抗体であってもよい。抗体フラグメントとしては、Ｆ（ａｂ’）２、Ｆ（ａｂ）２、Ｆａｂ’、Ｆａｂ、Ｆｖ、ｓｃＦｖなどが挙げられる。As used herein, the term "antibody" refers to a full-length immunoglobulin molecule that is naturally occurring or produced by genetic recombination technology, or an immunologically active fragment of an immunoglobulin molecule such as an antibody fragment. To say. These antibodies can be made using conventional techniques and may be polyclonal antibodies or monoclonal antibodies. Examples of the antibody fragment include F (ab') 2 , F (ab) 2 , Fab', Fab, Fv, scFv and the like.
本発明において使用する抗ＡｐｏＡ４抗体は、ＡｐｏＡ４タンパク質に特異的に結合する抗体であることが好ましい。「特異的な結合」とは、非特異的な相互作用に比して、より高い結合親和性でＡｐｏＡ４タンパク質と結合する抗体を指す。本発明の一実施形態では、「特異的な結合」は、例えばＡｐｏＡ４タンパク質に対して少なくとも約１０−４Ｍ、または少なくとも約１０−５Ｍ、または少なくとも約１０−６Ｍ、または少なくとも約１０−７Ｍ、または少なくとも約１０−８Ｍ、または少なくとも約１０−９Ｍ、または少なくとも約１０−１０Ｍ、または少なくとも約１０−１１Ｍ、または少なくとも約１０−１２Ｍ、またはそれ以上のＫｄを持つ抗体によって示されうる。別の実施形態では、「特異的な結合」は、ＡｐｏＡ４のポリペプチドとは異なる他のポリペプチドに実質的に結合することなく、ＡｐｏＡ４に結合することを意味する。The anti-ApoA4 antibody used in the present invention is preferably an antibody that specifically binds to the ApoA4 protein. "Specific binding" refers to an antibody that binds to the ApoA4 protein with a higher binding affinity compared to non-specific interactions. In one embodiment of the present invention, "specific binding", for example, at least about 10 -4 M against ApoA4 protein or at least about 10 -5 M, alternatively at least about 10 -6 M, or at least about 10, - Have Kd of 7 M, or at least about 10-8 M, or at least about 10-9 M, or at least about 10-10 M, or at least about 10-11 M, or at least about 10-12 M, or more. Can be indicated by antibody. In another embodiment, "specific binding" means binding to ApoA4 without substantially binding to another polypeptide different from that of ApoA4.
イムノアッセイは、検出可能に標識した抗ＡｐｏＡ４抗体、または、検出可能に標識した抗ＡｐｏＡ４抗体に対する抗体（二次抗体）を用いる。抗体の標識法により、エンザイムイムノアッセイ（ＥＩＡまたはＥＬＩＳＡ）、ラジオイムノアッセイ（ＲＩＡ）、蛍光イムノアッセイ（ＦＩＡ）、蛍光偏光イムノアッセイ（ＦＰＩＡ）、化学発光イムノアッセイ（ＣＬＩＡ）、電気化学発光イムノアッセイ（ＥＣＬＩＡ）等に分類され、これらのいずれも本発明の方法に用いることができる。 The immunoassay uses a detectably labeled anti-ApoA4 antibody or an antibody against a detectably labeled anti-ApoA4 antibody (secondary antibody). Classified into enzyme immunoassay (EIA or ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), fluorescence polarization immunoassay (FPIA), chemiluminescence immunoassay (CLIA), chemiluminescence immunoassay (ECLIA), etc. according to the antibody labeling method. Any of these can be used in the method of the present invention.
ＥＬＩＳＡ法では、ペルオキシダーゼ、アルカリホスファターゼ等の酵素、ＲＩＡ法では、１２５Ｉ、１３１Ｉ、３５Ｓ、３Ｈ等の放射性物質、ＦＰＩＡ法では、フルオレセインイソチオシアネート、ローダミン、ダンシルクロリド、フィコエリスリン、テトラメチルローダミンイソチオシアネート、近赤外蛍光材料等の蛍光物質、ＣＬＩＡ法では、ルシフェラーゼ、βガラクトシダーゼ等の酵素と各酵素で発光物質に変化する発光基質、ルシフェリン、エクオリン等の発光物質で標識した抗体を用いることができる。その他、金コロイド、量子ドットなどのナノ粒子で標識した抗体を検出することもできる。The ELISA method, peroxidase, an enzyme such as alkaline phosphatase, the RIA method, 125 I, 131 I, 35 S, 3 radioactive substances such as H, in the FPIA method, fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetra Fluorescent substances such as methylrhodamine isothiocyanate and near-infrared fluorescent materials, and in the CLIA method, enzymes labeled with enzymes such as luciferase and β-galactosidase, luminescent substrates that are converted into luminescent substances by each enzyme, and antibodies labeled with luminescent substances such as luciferin and equolin. Can be used. In addition, antibodies labeled with nanoparticles such as colloidal gold and quantum dots can be detected.
また、イムノアッセイでは、抗ＡｐｏＡ４抗体をビオチンで標識し、酵素等で標識したアビジンまたはストレプトアビジンを結合させて、ＡｐｏＡ４タンパク質を検出、測定することもできる。 Further, in the immunoassay, the anti-ApoA4 antibody can be labeled with biotin, and avidin or streptavidin labeled with an enzyme or the like can be bound to detect and measure the ApoA4 protein.
イムノアッセイの中でも、酵素標識を用いるＥＬＩＳＡ法は、簡便且つ迅速に標的を測定することができて好ましい。 Among the immunoassays, the ELISA method using an enzyme label is preferable because the target can be measured easily and quickly.
ＥＬＩＳＡ法では、例えばサンドイッチ法を用いることができる。固相担体に抗ＡｐｏＡ４抗体を固定し、適宜処理した生体試料を添加して反応させた後、さらに酵素で標識した別のエピトープを認識する抗ＡｐｏＡ４抗体を添加して反応させる。 In the ELISA method, for example, the sandwich method can be used. An anti-ApoA4 antibody is immobilized on a solid phase carrier, an appropriately treated biological sample is added and reacted, and then an anti-ApoA4 antibody that recognizes another enzyme-labeled epitope is added and reacted.
洗浄後、酵素基質と反応、発色させ、吸光度を測定することにより、ＡｐｏＡ４タンパク質の発現レベルを求めることができる。また、固相担体に固定した抗ＡｐｏＡ４抗体と生体試料中のＡｐｏＡ４タンパク質を反応させた後、非標識抗ＡｐｏＡ４抗体（一次抗体）を添加し、この非標識抗体に対する抗体（二次抗体）を酵素標識してさらに添加してもよい。 After washing, the expression level of ApoA4 protein can be determined by reacting with the enzyme substrate, developing a color, and measuring the absorbance. Further, after reacting the anti-ApoA4 antibody immobilized on the solid phase carrier with the ApoA4 protein in the biological sample, an unlabeled anti-ApoA4 antibody (primary antibody) is added, and an antibody (secondary antibody) against this unlabeled antibody is enzymed. It may be labeled and added further.
酵素基質は、酵素がペルオキシダーゼの場合、３，３’−ジアミノベンジン（３，３’−ｄｉａｍｉｎｏｂｅｎｚｉｄｉｎｅ；ＤＡＢ）、３，３’，５，５’−テトラメチルベンジン（３，３’，５，５’−ｔｅｔｒａｍｅｔｈｙｌｂｅｎｚｉｄｉｎｅ；ＴＭＢ）、ｏ−フェニレンジアミン（ｏ−ｐｈｅｎｙｌｅｎｅｄｉａｍｉｎｅ；ＯＰＤ）等を用いることができ、アルカリホスファターゼの場合、ｐ−ニトロフェニルホスフェート（ｐ−ｎｉｔｒｏｐｈｅｎｙ ｐｈｏｓｐｈａｔｅ；ＮＰＰ）等を用いることができる。 The enzyme substrate is 3,3'-diaminobenzine (DAB), 3,3', 5,5'-tetramethylbenzine (3,3', 5,5) when the enzyme is peroxidase. '-Tetramethylbenzidine; TMB), o-phenylenediamine (OPD) and the like can be used, and in the case of alkaline phosphatase, p-nitrophenyl phosphate (NPP) and the like can be used.
また、上記イムノアッセイの中で、微量のタンパク質を簡便に検出できる方法として凝集法も好ましい。凝集法としては、例えば、抗体にラテックス粒子を結合させたラテックス凝集法が挙げられる。 In addition, the agglutination method is also preferable as a method for easily detecting a trace amount of protein in the above immunoassay. Examples of the agglutination method include a latex agglutination method in which latex particles are bound to an antibody.
ラテックス粒子に抗ＡｐｏＡ４抗体を結合させて生体試料に混合すると、ＡｐｏＡ４タンパク質が存在すれば、抗体結合ラテックス粒子が凝集する。そこで、試料に近赤外光を照射して、吸光度の測定（比濁法）または散乱光の測定（比朧法）により凝集塊を定量し、抗原の濃度を求めることができる。 When the anti-ApoA4 antibody is bound to the latex particles and mixed with the biological sample, the antibody-bound latex particles aggregate in the presence of the ApoA4 protein. Therefore, the sample can be irradiated with near-infrared light, and the agglomerates can be quantified by measuring the absorbance (turbidity method) or the scattered light (the turbidity method) to determine the concentration of the antigen.
ＡｐｏＡ４遺伝子の発現レベルの測定は、ＡｐｏＡ４ ｍＲＮＡを定量的に検出できる方法であれば特に制限されず、例えば、ＡｐｏＡ４ ｍＲＮＡに特異的なプライマーを用いたＰＣＲ法、ＲＴ−ＰＣＲ法、リアルタイムＰＣＲ法などの核酸増幅法を用いることができる。例えば、リアルタイムＰＣＲは、具体的には、以下のように行うことができる。すなわち、組織からトータルＲＮＡを常法に従って取得し、逆転写酵素と適当なプライマーを用いてｃＤＮＡを作製する。作製したｃＤＮＡをもとに、ＡｐｏＡ４遺伝子に特異的なプライマーと、ＤＮＡポリメラーゼを用いることでＡｐｏＡ４遺伝子を特異的に増幅させることができる。ＡｐｏＡ４遺伝子は、増幅させる際に適当な蛍光色素を取り込ませることで、特定の機器（ＰＲＩＳＭ ７７００ Ｓｅｑｕｅｎｃｅ Ｄｅｔｅｃｔｏｒ ７９００ＨＴ Ｓｅｑｕｅｎｃｅ Ｄｅｔｅｃｔｉｏｎ ＳｙｓｔｅｍもしくはＶｉｉＡ（商標）７ Ｒｅａｌ−Ｔｉｍｅ ＰＣＲ Ｓｙｓｔｅｍ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）など）を用いて増幅曲線をモニターすることができる。内部標準遺伝子（Ｈｐｒｔなど）によりＡｐｏＡ４の補正を行うことで、組織におけるＡｐｏＡ４ ｍＲＮＡの量を定量的に測定することができる。 The measurement of the expression level of the ApoA4 gene is not particularly limited as long as it is a method capable of quantitatively detecting ApoA4 mRNA. For example, PCR method using primers specific for ApoA4 mRNA, RT-PCR method, real-time PCR method, etc. Nucleic acid amplification method can be used. For example, real-time PCR can be specifically performed as follows. That is, total RNA is obtained from a tissue according to a conventional method, and cDNA is prepared using reverse transcriptase and an appropriate primer. Based on the prepared cDNA, the ApoA4 gene can be specifically amplified by using a primer specific to the ApoA4 gene and a DNA polymerase. By incorporating an appropriate fluorescent dye when amplifying the ApoA4 gene, a specific device (PRISM 7700 Sequence Detector 7900HT Sequence Detection System or ViiA ™ 7 Real-Time PCR System), Applied Biosystems, etc. can be used. The amplification curve can be monitored using. By correcting ApoA4 with an internal standard gene (Hprt, etc.), the amount of ApoA4 mRNA in the tissue can be quantitatively measured.
別の態様において、本発明は、ＨＧＦの薬力学的作用を評価するためのキットに関する。本発明に係るキットは、ＡｐｏＡ４のレベルを測定するための手段を含んでおり、一実施形態において、そのような手段は、抗ＡｐｏＡ４抗体、ＡｐｏＡ４アプタマー、ＡｐｏＡ４タンパク質、その断片若しくはそれらを同位体標識したもの、またはＡｐｏＡ４遺伝子配列に相補的な部分配列からなるプライマー，プローブ若しくはそれらを色素で標識したものである。本発明のキットの好ましい実施形態において、抗ＡｐｏＡ４抗体は、ＡｐｏＡ４に特異的に結合する抗体であり、ＡｐｏＡ４アプタマーは、ＡｐｏＡ４タンパク質に特異的に結合するアプタマーである。 In another aspect, the invention relates to a kit for assessing the pharmacodynamic effects of HGF. The kit according to the invention includes means for measuring the level of ApoA4, and in one embodiment such means are anti-ApoA4 antibody, ApoA4 aptamer, ApoA4 protein, fragments thereof or isotopic labeling thereof. A primer, a probe, or a dye-labeled one consisting of a partial sequence complementary to the ApoA4 gene sequence. In a preferred embodiment of the kit of the invention, the anti-ApoA4 antibody is an antibody that specifically binds to ApoA4, and the ApoA4 aptamer is an aptamer that specifically binds to the ApoA4 protein.
本発明のキットは、ＡｐｏＡ４タンパク質と、抗ＡｐｏＡ４抗体との抗原抗体反応を利用するイムノアッセイによって、生体試料中のＡｐｏＡ４タンパク質の量を測定するために必要な試薬および装置をさらに含んでもよい。 The kit of the present invention may further include reagents and devices necessary to measure the amount of ApoA4 protein in a biological sample by immunoassay utilizing the antigen-antibody reaction between ApoA4 protein and anti-ApoA4 antibody.
本発明のキットは、一実施形態において、サンドイッチＥＬＩＳＡ法によってＡｐｏＡ４タンパク質の量を測定するためのものであり、マイクロタイタープレート；捕捉用の抗ＡｐｏＡ４抗体；アルカリホスファターゼまたはペルオキシダーゼで標識した抗ＡｐｏＡ４抗体；および、アルカリホスファターゼ基質（ＮＰＰ等）またはペルオキシダーゼの基質（ＤＡＢ、ＴＭＢ、マイクロタイタープレートＯＰＤ等）、を含む。捕捉抗体と標識抗体は、異なるエピトープを認識する。このようなキットの場合、まず、マイクロタイタープレートに捕捉抗体を固定し、ここに適宜処理し希釈した生体試料を添加した後インキュベートし、試料を除去して洗浄する。次に、標識抗体を添加した後インキュベートし、基質を加えて発色させる。マイクロタイタープレートリーダー等を用いて発色を測定することにより、ＡｐｏＡ４タンパク質の量を求めることができる。 The kit of the present invention is for measuring the amount of ApoA4 protein by the sandwich ELISA method in one embodiment; microtiter plate; anti-ApoA4 antibody for capture; anti-ApoA4 antibody labeled with alkaline phosphatase or peroxidase; And an alkaline phosphatase substrate (NPP, etc.) or a peroxidase substrate (DAB, TMB, microtiter plate OPD, etc.). The capture antibody and the labeled antibody recognize different epitopes. In the case of such a kit, first, the capture antibody is fixed on a microtiter plate, an appropriately treated and diluted biological sample is added thereto, and then the incubation is performed, and the sample is removed and washed. Next, the labeled antibody is added and then incubated, and the substrate is added to develop color. The amount of ApoA4 protein can be determined by measuring the color development using a microtiter plate reader or the like.
本発明のキットは、別の実施形態において、二次抗体を使用したサンドイッチＥＬＩＳＡ法によりＡｐｏＡ４タンパク質の量を測定するためのものであり、マイクロタイタープレート；捕捉用の抗ＡｐｏＡ４抗体；一次抗体として、抗ＡｐｏＡ４抗体；二次抗体として、アルカリホスファターゼまたはペルオキシダーゼで標識した、一次抗体の抗ＡｐｏＡ４抗体に対する抗体；および、アルカリホスファターゼ（ＮＰＰ等）またはペルオキシダーゼの基質（ＤＡＢ、ＴＭＢ、ＯＰＤ等）、を含む。捕捉抗体と一次抗体は、異なるエピトープを認識する。このようなキットでは、まず、マイクロタイタープレートに捕捉抗体を固定し、ここに適宜処理し希釈した生体試料を添加した後インキュベートし、試料を除去して洗浄する。続いて、一次抗体を添加してインキュベートおよび洗浄を行い、さらに酵素標識した二次抗体を添加してインキュベートを行った後、基質を加えて発色させる。マイクロタイタープレートリーダー等を用いて発色を測定することにより、ＡｐｏＡ４タンパク質の量を求めることができる。二次抗体を用いることにより、反応が増幅され検出感度を高めることができる。 In another embodiment, the kit of the present invention is for measuring the amount of ApoA4 protein by the sandwich ELISA method using a secondary antibody, as a microtiter plate; anti-ApoA4 antibody for capture; as a primary antibody. Anti-ApoA4 antibody; as a secondary antibody, an antibody against the anti-ApoA4 antibody of the primary antibody labeled with alkaline phosphatase or peroxidase; and an alkaline phosphatase (NPP, etc.) or peroxidase substrate (DAB, TMB, OPD, etc.). The capture antibody and the primary antibody recognize different epitopes. In such a kit, first, the capture antibody is immobilized on a microtiter plate, an appropriately treated and diluted biological sample is added thereto, and then the sample is incubated, and the sample is removed and washed. Subsequently, the primary antibody is added for incubation and washing, and the enzyme-labeled secondary antibody is further added for incubation, and then a substrate is added for color development. The amount of ApoA4 protein can be determined by measuring the color development using a microtiter plate reader or the like. By using the secondary antibody, the reaction can be amplified and the detection sensitivity can be increased.
本発明のキットは、さらに、必要な緩衝液、酵素反応停止液、マイクロプレートリーダー、製品説明書等を含むことも好ましい。 It is also preferable that the kit of the present invention further contains the necessary buffer solution, enzyme reaction terminator solution, microplate reader, product description and the like.
標識抗体は、酵素標識した抗体に限定されず、放射性物質（１２５Ｉ、１３１Ｉ、３５Ｓ、３Ｈ等）、蛍光物質（フルオレセインイソチオシアネート、ローダミン、ダンシルクロリド、フィコエリスリン、テトラメチルローダミンイソチオシアネート、近赤外蛍光材料等）、発光物質（ルシフェラーゼ、ルシフェリン、エクオリン等）、ナノ粒子（金コロイド、量子ドット）等で標識した抗体であってもよい。また標識抗体としてビオチン化抗体を用い、キットに標識したアビジンまたはストレプトアビジンを加えることもできる。Labeled antibody is not limited to antibodies labeled with an enzyme, radioactive (125 I, 131 I, 35 S, 3 H , etc.), fluorescent substance (fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine iso Antibodies labeled with thiocyanates, near-infrared fluorescent materials, etc.), luminescent substances (luciferase, luciferin, equolin, etc.), nanoparticles (gold colloid, quantum dots, etc.) may be used. It is also possible to use a biotinylated antibody as a labeled antibody and add avidin or streptavidin labeled in the kit.
本発明のキットは、ＡｐｏＡ４と、ＡｐｏＡ４アプタマーとの反応を利用するアッセイによって、生体試料中のＡｐｏＡ４の量を測定するために必要な試薬および装置をさらに含んでもよい。 The kit of the present invention may further include the reagents and equipment required to measure the amount of ApoA4 in a biological sample by an assay utilizing the reaction of ApoA4 with an ApoA4 aptamer.
なお、本明細書において用いられる用語は、特定の実施形態を説明するために用いられるのであり、発明を限定する意図ではない。 It should be noted that the terms used in the present specification are used to explain a specific embodiment, and are not intended to limit the invention.
また、本明細書において用いられる「含む」との用語は、文脈上明らかに異なる理解をすべき場合を除き、記述された事項（部材、ステップ、要素、数字など）が存在することを意図するものであり、それ以外の事項（部材、ステップ、要素、数字など）が存在することを排除しない。 Also, as used herein, the term "contains" is intended to include the matters described (members, steps, elements, numbers, etc.) unless the context clearly requires a different understanding. It does not exclude the existence of other matters (members, steps, elements, numbers, etc.).
異なる定義が無い限り、ここに用いられるすべての用語（技術用語および科学用語を含む。）は、本発明が属する技術の当業者によって広く理解されるのと同じ意味を有する。ここに用いられる用語は、異なる定義が明示されていない限り、本明細書および関連技術分野における意味と整合的な意味を有するものとして解釈されるべきであり、理想化され、または、過度に形式的な意味において解釈されるべきではない。 Unless defined differently, all terms used herein (including technical and scientific terms) have the same meaning as widely understood by those skilled in the art to which the present invention belongs. The terms used herein should be construed as having a meaning consistent with that in the specification and related arts, unless otherwise defined, idealized or over-formed. Should not be interpreted in a positive sense.
第一の、第二のなどの用語が種々の要素を表現するために用いられる場合があるが、これらの要素はそれらの用語によって限定されるべきではないことが理解される。これらの用語は一つの要素を他の要素と区別するためのみに用いられているのであり、例えば、第一の要素を第二の要素と記し、同様に、第二の要素は第一の要素と記すことは、本発明の範囲を逸脱することなく可能である。 It is understood that terms such as first, second, etc. may be used to describe various elements, but these elements should not be limited by those terms. These terms are used only to distinguish one element from the other, for example, the first element is referred to as the second element, and similarly, the second element is the first element. Is possible without departing from the scope of the present invention.
以下、実施例により本発明をより具体的に説明するが、しかしながら、本発明はいろいろな形態により具現化することができ、ここに記載される実施例に限定されるものとして解釈されてはならない。 Hereinafter, the present invention will be described in more detail with reference to Examples, however, the present invention can be embodied in various forms and should not be construed as being limited to the examples described herein. ..
［実施例１］ｒｈ−ＨＧＦを投与した健常マウスの肝組織を用いた遺伝子発現解析
目的：
ｒｈ−ＨＧＦが生物活性をもたらす標的臓器、肝臓を材料として、ｒｈ−ＨＧＦによって影響を受ける分子を同定し、その中からＰＤマーカーの同定を試みた。分子同定には、マイクロアレイならびにリアルタイムＰＣＲ法を用いた。[Example 1] Gene expression analysis using liver tissue of healthy mice administered with rh-HGF
Purpose:
Using the target organ and liver where rh-HGF brings about biological activity as materials, we identified molecules affected by rh-HGF and attempted to identify PD markers from them. Microarray and real-time PCR method were used for molecular identification.
実験：
ｒｈ−ＨＧＦの投与と採材
チャイニーズハムスター卵巣（ＣＨＯ）細胞で組み換え発現させた肝細胞増殖因子アクチベーターを用いて、ＣＨＯ細胞で組み換え発現させたヒトＨＧＦを活性化して、ｒｈ−ＨＧＦを得た。ＢＡＬＢ／ｃＡｎＮＣｒｌＣｒｌｊ雄性６週齢マウス（日本チャールスリバー）に、ダルベツコリン酸緩衝生理食塩水（Ｄｕｌｂｅｃｃｏ‘ｓ Ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ：ＰＢＳ）（Ｗａｋｏ，♯０４５−２９７９５）またはｒｈ−ＨＧＦ（１．５ｍｇ／ｋｇ）を静脈内投与した。ＰＢＳ投与直後、ｒｈ−ＨＧＦの投与後８および２４時間に、３％イソフルラン吸入麻酔下にて腹部下大静脈より採血を行い、頚椎脱臼法によりマウスを安楽死させた。安楽死後、肝臓組織を採取し、ＲＮＡ ｌａｔｅｒ（Ａｍｂｉｏｎ， ＃ＡＭ７０２１）に浸漬させた。下記表は、本実験に用いた試験群を示す。 Experiment:
Administration of rh-HGF and collection Using a hepatocyte growth factor activator recombinantly expressed in Chinese hamster ovary (CHO) cells, human HGF recombinantly expressed in CHO cells was activated to obtain rh-HGF. .. BALB / cAnNCrlCrlj male 6-week-old mice (Charles River, Japan) with Dulbecco's Phosphate Buffered Saline (PBS) (Wako, # 045-29795) or rh-HGF (1.5 mg / kg) Was administered intravenously. Immediately after the administration of PBS, 8 and 24 hours after the administration of rh-HGF, blood was collected from the inferior abdominal vein under 3% isoflurane inhalation anesthesia, and the mice were euthanized by cervical dislocation. After euthanasia, liver tissue was harvested and immersed in RNA lattice (Ambion, # AM7021). The table below shows the test groups used in this experiment.
肝臓由来Ｔｏｔａｌ ＲＮＡの抽出
採材した肝臓からＴＲＩＺＯＬ Ｒｅａｇｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ， ＃１５５９６−０１８）、ＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ（ＱＩＡＧＥＮ， ＃７４１０４）、およびＲＮａｓｅ−Ｆｒｅｅ ＤＮａｓｅ ｓｅｔ（ＱＩＡＧＥＮ， ＃７９２５４）を用いてｔｏｔａｌ ＲＮＡを精製した。ＲＮＡ濃度を吸光度により測定した後、ＲＮＡ ６０００ Ｎａｎｏ Ａｓｓａｙ Ｋｉｔ（Ａｇｉｌｅｎｔ Ｔｅｃｈｎｏｌｏｇｉｅｓ， ＃５０６７−１５１１）を用いて、２１００ Ｂｉｏａｎａｌｙｚｅｒ（Ａｇｉｌｅｎｔ Ｔｅｃｈｎｏｌｏｇｉｅｓ）にてクオリティチェックを実施した。 Extraction of liver-derived total RNA Using TRIZOL Reagent (Invitrogen, # 15596-018), RNeasy Mini Kit (QIAGEN, # 74104), and RNase-Free DNase set (QIAGEN, # 79254) from the collected liver. Purified. After measuring the RNA concentration by absorbance, a quality check was performed on a 2100 Bioanalyzer (Agilent Technologies) using an RNA 6000 Nano Assay Kit (Agilent Technologies, # 5067-1511).
ＤＮＡマイクロアレイ解析
ＤＮＡマイクロアレイは、ＳｕｒｅＰｒｉｎｔ Ｇ３ ｍｏｕｓｅ ＧＥ ８×６０Ｋ（Ａｇｉｌｅｎｔ Ｔｅｃｈｎｏｌｏｇｉｅｓ， ＃Ｇ４８５２Ａ）を使用した。得られたデータのうち、３群全てでシグナル値が１００以上となっている遺伝子を対象とし、群１に対して群２または群３で２倍以上のシグナル値が増加したものを一時抽出した。その中から、可溶性タンパク質として存在しうると思われうる分子として、Ｇｅｎｅ Ｏｎｔｏｌｏｇｙ ａｎｎｏｔａｔｉｏｎで「Ｅｘｔｒａｃｅｌｌｕｌａｒ」あるいは「Ｐｌａｓｍａ ｍｅｍｂｒａｎｅ」の情報を有するものを抽出した。このようにして、ｒｈ−ＨＧＦ投与によって発現が強く誘導される分子をＰＤマーカー候補として選んだ。 DNA Microarray Analysis As the DNA microarray, a SurePrint G3 mouse GE 8 × 60K (Agilent Technologies, # G4852A) was used. Among the obtained data, genes having a signal value of 100 or more in all three groups were targeted, and those in which the signal value increased more than twice in group 2 or 3 with respect to group 1 were temporarily extracted. .. Among them, as a molecule that may exist as a soluble protein, a molecule having information on "Extracellular" or "Plasma membrane" was extracted by Gene Ontology annotation. In this way, molecules whose expression is strongly induced by rh-HGF administration were selected as PD marker candidates.
結果１：
上記の分子選出法によって、ＡｐｏＡ４分子を抽出した（表２）。ＡｐｏＡ４分子は、アポリポタンパク質を構成するアポタンパク質として血中に存在する分子である。ＡｐｏＡ４分子は、ＰＢＳのみを投与されたマウス（群１）では発現が低く、ｒｈ−ＨＧＦを投与すると発現増強が見られた。 Result 1:
ApoA4 molecules were extracted by the above molecular selection method (Table 2). The ApoA4 molecule is a molecule that exists in the blood as an apoprotein that constitutes an apolipoprotein. The expression of ApoA4 molecule was low in mice (Group 1) to which PBS alone was administered, and increased expression was observed when rh-HGF was administered.
リアルタイムＰＣＲによるＡｐｏＡ４ ｍＲＮＡ発現誘導の確定
ＤＮＡマイクロアレイで用いたサンプルと同じＲＮＡについて、Ｈｉｇｈ−Ｃａｐａｃｉｔｙ ｃＤＮＡ Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔｉｏｎ Ｋｉｔ ｗｉｔｈ ＲＮａｓｅ Ｉｎｈｉｂｉｔｏｒ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ， ＃４３７４９６６）を使用して逆転写し、ｃＤＮＡを作製した。得られたｃＤＮＡを鋳型として、ＡｐｏＡ４ ｍＲＮＡ発現量をリアルタイムＰＣＲ法で検討した。検討にあたっては、Ｈｐｒｔ ｍＲＮＡを内部コントロールとした。ＰＣＲ反応は、ＴａｑＭａｎ Ｆａｓｔ Ａｄｖａｎｃｅｄ Ｍａｓｔｅｒ Ｍｉｘ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ， ＃４４４４５５７）とＴａｑＭａｎ Ａｓｓａｙ（ＡｐｏＡ４： Ｍｍ００４３１８１４＿ｍ１， Ｈｐｒｔ： Ｍｍ０３０２４０７５＿ｍ１， Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いて、ＶｉｉＡ７リアルタイムＰＣＲシステム（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を使用して、ＰＣＲの増幅反応を実施した。増幅反応は、９５℃で２０秒インキュベートした後、９５℃で１秒、６０℃で２０秒の反応を４０サイクル繰り返した。ＡｐｏＡ４ ｍＲＮＡの発現値をサンプルごとにＨｐｒｔ ｍＲＮＡ発現値で標準化することで、サンプル間の補正を実施した。 The same RNA as the sample used in the confirmed DNA microarray for induction of ApoA4 mRNA expression by real-time PCR was reverse transcribed using the High-Capacity cDNA Reverse Transcript Kit with RNase Inhibitor (Applied Biosystems, # 4374966). Using the obtained cDNA as a template, the expression level of ApoA4 mRNA was examined by a real-time PCR method. In the study, Hprt mRNA was used as an internal control. PCR reaction is performed using TaqMan Fast Advanced Master Mix (Applied Biosystems, # 4444557) and TaqMan Assay (ApoA4: Mm00431814_m1, HPrt: Mm03024075_Mm1, An amplification reaction was carried out. In the amplification reaction, after incubating at 95 ° C. for 20 seconds, the reaction at 95 ° C. for 1 second and 60 ° C. for 20 seconds was repeated for 40 cycles. Correction between samples was performed by standardizing the expression value of ApoA4 mRNA with the expression value of Hprt mRNA for each sample.
統計解析：
ＡｐｏＡ４ ｍＲＮＡ定量の結果について、コントロールとｒｈ−ＨＧＦ投与群との間で、ダネットの多重比較検定を実施した（図１）。 Statistical analysis:
For the results of ApoA4 mRNA quantification, a multiple comparison test of Danette was performed between the control and the rh-HGF-administered group (Fig. 1).
結果２：
ＤＮＡマイクロアレイの結果得られたＡｐｏＡ４について、そのｍＲＮＡをリアルタイムＰＣＲ法で定量し確定した。ハウスキーピング遺伝子のＨｐｒｔ ｍＲＮＡを内部コントロールとしたＡｐｏＡ４ ｍＲＮＡ定量の結果、その挙動はＤＮＡマイクロアレイで見られたシグナル値の変動と相関しており、ｒｈ−ＨＧＦ投与によって強く発現が増強されていることが確認された（図１）。 Result 2:
The mRNA of ApoA4 obtained as a result of the DNA microarray was quantified and confirmed by the real-time PCR method. As a result of quantification of ApoA4 mRNA using Hprt mRNA of the housekeeping gene as an internal control, its behavior correlates with the fluctuation of the signal value observed in the DNA microarray, and it is found that the expression is strongly enhanced by the administration of rh-HGF. It was confirmed (Fig. 1).
［実施例２］ｒｈ−ＨＧＦ投与によるＡｐｏＡ４誘導とｃ−Ｍｅｔ阻害剤によるキャンセル効果
目的：
ｒｈ−ＨＧＦの生物活性は、その受容体ｃ−Ｍｅｔを介してもたらされる。即ち、ＡｐｏＡ４がｒｈ−ＨＧＦのＰＤマーカーであることの妥当性は、その発現誘導がｃ−Ｍｅｔに依存していることを明らかにすることによって担保できる。そこで、ｃ−Ｍｅｔの特異的阻害剤を投与した健常マウスを用いて、ｒｈ−ＨＧＦ投与によるＡｐｏＡ４発現誘導のキャンセル効果を検討した。検討は肝臓由来ｍＲＮＡおよび血清中タンパク質を対象とした。[Example 2] ApoA4 induction by rh-HGF administration and cancellation effect by c-Met inhibitor
Purpose:
The biological activity of rh-HGF is mediated by its receptor c-Met. That is, the validity of ApoA4 as a PD marker for rh-HGF can be ensured by clarifying that its expression induction depends on c-Met. Therefore, the effect of canceling the induction of ApoA4 expression by rh-HGF administration was examined using healthy mice administered with a specific inhibitor of c-Met. The study targeted liver-derived mRNA and serum proteins.
実験：
ｃ−Ｍｅｔ阻害剤の投与と実験群
ＢＡＬＢ／ｃＡｎＮＣｒｌＣｒｌｊ雄性６週齢マウス（日本チャールスリバー）にｃ−Ｍｅｔ阻害剤ＰＦ−０４２１７９０３（５０ ｍｇ／ｋｇ）（Ｓｅｌｌｅｃｋ Ｃｈｅｍｉｃａｌｓ ＃Ｓ１０９４， ｌｏｔ． ０５）または媒体（５％ ＤＭＳＯ，１０％ Ｔｗｅｅｎ８０，６．７ｍｍｏｌ／Ｌ塩酸水）を１０ｍＬ／ｋｇの用量で経口投与し、１時間後にｒｈ−ＨＧＦ（０．５， １．５， ５．０， １５ｍｇ／ｋｇ）またはＰＢＳを静脈内投与した。ｒｈ−ＨＧＦまたはＰＢＳの投与後２４時間、３％イソフルラン吸入麻酔下にて腹部下大静脈より採血を行い、頚椎脱臼法によりマウスを安楽死させた。安楽死後、肝臓組織を採取し、ＲＮＡ ｌａｔｅｒ（Ａｍｂｉｏｎ，＃ＡＭ７０２１）に浸漬させた。採取した血液から血清を回収した。下記表は、本実験で用いた試験群を示す。 Experiment:
Administration of c-Met inhibitor and experimental group BALB / cAnNCrlCrlj male 6-week-old mice (Charles River, Japan) c-Met inhibitor PF-04217903 (50 mg / kg) (Selleck Chemicals # S1094, lot. 05) or medium (5% DMSO, 10% Tween 80, 6.7 mmol / L hydrochloric acid) was orally administered at a dose of 10 mL / kg, and 1 hour later, rh-HGF (0.5, 1.5, 5.0, 15 mg / kg). ) Or PBS was administered intravenously. Blood was collected from the inferior abdominal vein under 3% isoflurane inhalation anesthesia for 24 hours after administration of rh-HGF or PBS, and the mice were euthanized by cervical dislocation. After euthanasia, liver tissue was harvested and immersed in RNA lattice (Ambion, # AM7021). Serum was collected from the collected blood. The table below shows the test groups used in this experiment.
肝臓由来Ｔｏｔａｌ ＲＮＡの抽出
採材した肝臓からＴＲＩＺＯＬ Ｒｅａｇｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ， ＃１５５９６−０１８）、ＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ（ＱＩＡＧＥＮ， ＃７４１０４）、およびＲＮａｓｅ−Ｆｒｅｅ ＤＮａｓｅ ｓｅｔ（ＱＩＡＧＥＮ， ＃７９２５４）を用いてｔｏｔａｌ ＲＮＡを精製し、ＲＮＡ濃度を吸光度により測定した。 Extraction of liver-derived total RNA Using TRIZOL Reagent (Invitrogen, # 15596-018), RNeasy Mini Kit (QIAGEN, # 74104), and RNase-Free DNase set (QIAGEN, # 79254) from the collected liver. After purification, RNA concentration was measured by absorbance.
リアルタイムＰＣＲ法によるＡｐｏａ４ ｍＲＮＡの定量
リアルタイムＰＣＲ法によるＡｐｏＡ４ ｍＲＮＡの定量は、実施例１に準じて行った。 Quantification of Apoa4 mRNA by real-time PCR method The quantification of ApoA4 mRNA by real-time PCR method was performed according to Example 1.
統計解析：
ＡｐｏＡ４ ｍＲＮＡ定量の結果について、コントロールとｒｈ−ＨＧＦ投与群との間で、ダネットの多重比較検定を実施した。さらに、ＡｐｏＡ４ ｍＲＮＡが統計的に有意に増加したｒｈ−ＨＧＦ処置群について、ビヒクルとｃ−Ｍｅｔ阻害剤処置群との間で対応のないｔ検定を実施した（図２）。 Statistical analysis:
For the results of ApoA4 mRNA quantification, a multiple comparison test of Danette was performed between the control and the rh-HGF-administered group. Furthermore, for the rh-HGF-treated group in which ApoA4 mRNA was statistically significantly increased, an unpaired t-test was performed between the vehicle and the c-Met inhibitor-treated group (Fig. 2).
結果１：
ＡｐｏＡ４ ｍＲＮＡはｒｈ−ＨＧＦ投与により肝臓で顕著に発現が誘導・亢進することが再度確認された。更に、その誘導は、ｃ−Ｍｅｔ阻害剤を投与することで顕著に阻害されたことから、ｒｈ−ＨＧＦ投与に伴うＡｐｏＡ４ ｍＲＮＡの発現増強は、ｃ−Ｍｅｔ依存性であることが確認できた（図２）。 Result 1:
It was reconfirmed that the expression of ApoA4 mRNA was remarkably induced and enhanced in the liver by administration of rh-HGF. Furthermore, the induction was significantly inhibited by administration of a c-Met inhibitor, confirming that the enhancement of ApoA4 mRNA expression associated with rh-HGF administration was c-Met-dependent (). Figure 2).
血清ＡｐｏＡ４タンパク質の定量
上記血清をもちいて、ウェスタンブロット法によってＡｐｏＡ４タンパク質を定量した。ＰＢＳ（−）（Ｗａｋｏ）で２００倍希釈した血清に１／４容の泳動バッファー液（３１３ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８），１０％ ＳＤＳ，３０％ Ｇｌｙｃｅｒｏｌ，０．２ｍｇ／ｍＬ Ｂｒｏｍｏ−ｐｈｅｎｏｌ ｂｌｕｅ，２５％ ２−Ｍｅｒｃａｐｔｏｅｔｈｎｏｌ）を添加し（血清の最終希釈度は２５０倍となる）、９５℃、３分加熱処理した。その内１２．５μＬをＳＤＳ−ＰＡＧＥに供し、ニトロセルロースフィルター（Ｓｃｈｌｅｉｃｈｅｒ＆Ｓｃｈｕｅｌｌ， ＢＡ８５）にブロッティングした。フィルターをブロッキング液（５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５），１５０ｍＭ ＮａＣｌ，５％ ｓｋｉｍ−ｍｉｌｋ，０．０１％ Ｔｗｅｅｎ２０，０．１％ ＮａＮ３）に浸し、室温で１時間振とうしてからブロッキング液を廃棄し、ｇｏａｔ ａｎｔｉ−ｍｏｕｓｅ Ａｐｏｌｉｐｏｐｒｏｔｅｉｎ Ａ−ＩＶ抗体（ＧｅｎｅＴｅｘ， ＃ＧＴＸ８８５３３）を０．４２μｇ／ｍＬ濃度で添加した反応溶液（５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５），１５０ｍＭ ＮａＣｌ，５％ ｓｋｉｍ−ｍｉｌｋ，４％ ＰＥＧ６０００，０．２％ ＥＤＴＡ／３Ｎａ，０．０１％ Ｔｗｅｅｎ２０，０．２％ Ｐｒｏｃｌｉｎ１５０）に浸し、室温下で２時間振とうした。洗浄液（５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５），１５０ｍＭ ＮａＣｌ，０．０１％ Ｔｗｅｅｎ２０）で３回洗浄した後、二次抗体のＨＲＰ標識ｒａｂｂｉｔ ａｎｔｉ−ｇｏａｔ ＩｇＧ（Ｈ＋Ｌ）（Ｉｎｖｉｔｒｏｇｅｎ）を１／２０００濃度で添加した上記反応溶液に浸し、室温で１時間振とうした。上記洗浄液で３回洗浄した後、化学発光溶液ＳｕｐｅｒＳｉｇｎａｌ Ｗｅｓｔ Ｄｕｒａ Ｅｘｔｅｎｄｅｄ Ｄｕｒａｔｉｏｎ Ｓｕｂｓｔｒａｔｅ（Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ）を加えて発光させ、ＬＡＳ−４０００（ＦＵＪＩＦＩＬＭ）でＡｐｏＡ４タンパク質を検出した。 Quantification of Serum ApoA4 Protein Using the above serum, ApoA4 protein was quantified by Western blotting. 1/4 volume of migration buffer (313 mM Tris-HCl (pH 6.8), 10% SDS, 30% Glycerol, 0.2 mg / mL Bromo-phenyl blue) in serum diluted 200-fold with PBS (-) (Wako) , 25% 2-Mercaptoethnor) was added (the final dilution of serum was 250 times), and heat treatment was performed at 95 ° C. for 3 minutes. Of this, 12.5 μL was subjected to SDS-PAGE and blotting was performed on a nitrocellulose filter (Schleicher & Schuell, BA85). Immerse the filter in blocking solution (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% skim-milk, 0.01% Tween 20, 0.1% NaN3), shake at room temperature for 1 hour, and then the blocking solution. The reaction solution (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% skim-milk) was added at a concentration of 0.42 μg / mL with the goat anti-mouse Apolipoprotin A-IV antibody (GeneTex, #GTX88533). , 4% PEG6000, 0.2% EDTA / 3Na, 0.01% Tween20, 0.2% Proclin150) and shaken at room temperature for 2 hours. After washing 3 times with a washing solution (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.01% Room 20), the secondary antibody HRP-labeled rabbit anti-goat IgG (H + L) (Invitrogen) was concentrated at 1/2000 concentration. It was immersed in the above reaction solution added in 1 and shaken at room temperature for 1 hour. After washing with the above-mentioned washing solution three times, the chemiluminescent solution SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) was added to cause light emission, and ApoA4 protein was detected by LAS-4000 (FUJIFILM).
統計解析：
血清ＡｐｏＡ４ タンパク質定量の結果について、コントロールとｒｈ−ＨＧＦ投与群との間で、ダネットの多重比較検定を実施した。さらに、ＡｐｏＡ４ タンパク質が統計的に有意に増加したｒｈ−ＨＧＦ処置群について、ビヒクル処置群とｃ−Ｍｅｔ阻害剤処置群との間で対応のないｔ検定を実施した（図３）。 Statistical analysis:
For the results of serum ApoA4 protein quantification, a multiple comparison test of Danette was performed between the control and the rh-HGF-administered group. In addition, an unpaired t-test was performed between the vehicle-treated group and the c-Met inhibitor-treated group for the rh-HGF-treated group in which the ApoA4 protein was statistically significantly increased (Fig. 3).
結果２：
ｒｈ−ＨＧＦ投与によるＡｐｏＡ４タンパク質の発現誘導は、血清中のタンパク質として検出することが可能だった。更に、その誘導は、ｃ−Ｍｅｔ阻害剤を投与することで阻害されたことから、ｒｈ−ＨＧＦ投与に伴うＡｐｏＡ４ タンパク質の発現増強は、ｃ−Ｍｅｔ依存性であることが確認できた（図３）。 Result 2:
Induction of ApoA4 protein expression by rh-HGF administration could be detected as a protein in serum. Furthermore, since the induction was inhibited by administration of a c-Met inhibitor, it was confirmed that the enhancement of ApoA4 protein expression associated with rh-HGF administration was c-Met-dependent (FIG. 3). ).
［実施例３］実験的肝障害時でのｒｈ−ＨＧＦ投与によるＡｐｏＡ４タンパク質の産生増強効果の検証
目的：
これまでの結果から、マウスＡｐｏＡ４タンパク質は血中で測定可能なＨＧＦのＰＤマーカーと考えられた。健常マウスの肝ｍＲＮＡならびに血中において、ＡｐｏＡ４タンパク質はＨＧＦのＰＤマーカーとしての挙動をしめしたが、肝障害時においてもＨＧＦのＰＤマーカーとしての挙動を示すか否かについて、実験的肝障害マウス（Ｊｏ２肝不全モデル）を用いて検証した。Ｊｏ２肝不全モデルは、抗Ｆａｓモノクローナル抗体（Ａｎｔｉ−Ｆａｓ ＭＡｂ）投与によって直接に肝細胞のアポトーシスを惹起することに起因した重症度の高い肝障害モデルである。[Example 3] Verification of the effect of enhancing the production of ApoA4 protein by administration of rh-HGF in the case of experimental liver injury
Purpose:
From the results so far, the mouse ApoA4 protein was considered to be a PD marker of HGF that can be measured in blood. In the liver mRNA and blood of healthy mice, the ApoA4 protein behaved as a PD marker for HGF, but whether or not it behaved as a PD marker for HGF even during liver failure was investigated in experimental liver-damaged mice ( It was verified using the Jo2 liver failure model). The Jo2 liver failure model is a highly severe liver injury model caused by directly inducing hepatocyte apoptosis by administration of an anti-Fas monoclonal antibody (Anti-Fas MAb).
実験：
Ｊｏ２肝不全モデルの作製
ＢＡＬＢ／ｃＡｎＮＣｒｌＣｒｌｊ雄性６週齢マウス（日本チャールスリバー）に、抗Ｆａｓモノクローナル抗体（クローン名：Ｊｏ２、ＢＤ Ｐｈａｒｍｉｎｇｅｎ， ＃５５４２５４， ｌｏｔ＃３２６９６３４）０．２３ｍｇ／ｋｇを単回尾静脈内投与し、急性肝不全モデルを作製した。このモデルに対し、ＰＢＳに溶解したｒｈ−ＨＧＦを０．１５， １．５， １５ ｍｇ／ｋｇで、抗Ｆａｓモノクローナル抗体投与の１時間前に尾静脈内投与した。コントロール群（Ｃｏｎｔｒｏｌ）には、ＰＢＳを尾静脈内投与した。 Experiment:
Preparation of Jo2 liver failure model BALB / cAnNCrlCrlj male 6-week-old mouse (Charles River Japan) was given 0.23 mg / kg of anti-Fas monoclonal antibody (clone name: Jo2, BD Harmingen, # 554254, lot # 3269634) once. Intravenous administration was performed to prepare a model of acute liver failure. For this model, rh-HGF dissolved in PBS was administered intravenously at 0.15, 1.5, 15 mg / kg 1 hour before administration of the anti-Fas monoclonal antibody. PBS was intravenously administered to the control group (Control).
肝障害の重症度評価
抗Ｆａｓモノクローナル抗体投与５時間後に３％イソフルラン吸入麻酔下にてマウスを保定し、顎骨後ろに位置する静脈より血液を採取し、コアグチェックＸＳ（Ｒｏｃｈｅ−ｄｉａｇｎｏｓｔｉｃｓ， ＃ＥＳ３５０００９）およびＰＴテストストリップ（Ｒｏｃｈｅ−ｄｉａｇｎｏｓｔｉｃｓ， ＃ＥＳ３５００５４）を用いてプロトロンビン時間（Ｐｒｏｔｈｒｏｍｂｉｎ ｔｉｍｅ：ＰＴ）を測定した。プロトロンビン時間測定後、腹部下大静脈より採血を行った後、頚椎脱臼法により安楽死させた。血清中のＡｓｐａｒｔａｔｅ Ａｍｉｎｏｔｒａｎｓｆｅｒａｓｅ（ＡＳＴ）、Ａｌａｎｉｎｅ ｔｒａｎｓａｍｉｎａｓｅ（ＡＬＴ）の測定は、それぞれＬタイプワコー ＡＳＴ・Ｊ２（Ｗａｋｏ）のキットおよびＬタイプワコー ＡＬＴ・Ｊ２（Ｗａｋｏ）のキットおよび自動分析装置（Ｈｉｔａｃｈｉ ７１８０ Ａｕｔｏｍａｔｉｃ Ａｎａｌｙｚｅｒ， Ｈｉｔａｃｈｉ Ｈｉｇｈ−Ｔｅｃｈｎｏｌｏｇｉｅｓ）を用いて行った。 Severity assessment of liver damage 5 hours after administration of anti-Fas monoclonal antibody, mice were retained under 3% isoflurane inhalation anesthesia, blood was collected from a vein located behind the jawbone, and coagcheck XS (Roche-diagnotics, # ES3500 09) was collected. ) And a PT test strip (Roche-diagnostics, # ES3500 54) were used to measure prothrombin time (PT). After measuring the prothrombin time, blood was collected from the inferior vena cava and then euthanized by cervical dislocation. Aspartate Aminotransphase (AST) and Alanine transaminase (ALT) in serum were measured by L-type Wako AST / J2 (Wako) kit and L-type Wako ALT / J2 (Wako) kit and automatic analyzer (Hitachi 7180), respectively. Analyzer, Hitachi High-Technologies) was used.
血清ＡｐｏＡ４タンパク質の定量
血清ＡｐｏＡ４タンパク質の定量は、実施例２に準じて行った。 Quantification of Serum ApoA4 Protein The quantification of serum ApoA4 protein was performed according to Example 2.
統計解析：
ＰＴ、ＡＳＴ、ＡＬＴおよび血清ＡｐｏＡ４タンパク質の測定の結果について、コントロールとｒｈ−ＨＧＦ投与群との間でダネットの多重比較検定を実施した（図４）。 Statistical analysis:
For the results of measurement of PT, AST, ALT and serum ApoA4 protein, a multiple comparison test of Danette was performed between the control and the rh-HGF-administered group (Fig. 4).
結果：
Ｊｏ２肝不全マウスにおいて、コントロールでは顕著な血中ＡＳＴ、ＡＬＴの上昇とＰＴの延長を認める一方、それらはｒｈ−ＨＧＦ投与によって強く阻害された。その時、血中ＡｐｏＡ４タンパク質の値は、ｒｈ−ＨＧＦの投与によって上昇し、１．５，５，１５ ｍｇ／ｋｇでは統計学的に有意な上昇を示した。以上から、ＨＧＦによる肝障害阻止効果を反映してＡｐｏＡ４タンパク質の上昇がみられることが判明した（図４）。 result:
In Jo2 liver failure mice, controls showed marked elevations in blood AST and ALT and prolongation of PT, while they were strongly inhibited by rh-HGF administration. At that time, the blood ApoA4 protein level was increased by administration of rh-HGF, and showed a statistically significant increase at 1.5, 5, 15 mg / kg. From the above, it was found that an increase in ApoA4 protein was observed reflecting the effect of HGF on preventing liver damage (Fig. 4).
［実施例４］ヒト初代培養肝細胞を用いたＡｐｏＡ４タンパク質のＰＤマーカーとしての検証
目的：
マウスを用いた実施例を通じて、血中ＡｐｏＡ４タンパク質がＨＧＦのＰＤマーカーであることを確認した。実際に、ヒトにおいてもＡｐｏＡ４タンパク質が、ＨＧＦのＰＤマーカーとなりうるのか否かについて、ヒト初代培養肝細胞を用いて検証した。[Example 4] Verification of ApoA4 protein as a PD marker using human primary cultured hepatocytes
Purpose:
Through the examples using mice, it was confirmed that the ApoA4 protein in blood was a PD marker for HGF. In fact, whether or not the ApoA4 protein can be a PD marker for HGF in humans was examined using human primary cultured hepatocytes.
実験：
ヒト初代培養肝細胞とｒｈ−ＨＧＦによる処理
２４ウェルプレートに播種したヒト初代培養肝細胞（３．８×１０５細胞／ウェル， Ｂｉｏｐｒｅｄｉｃ Ｉｎｔｅｒｎａｔｉｏｎａｌ， ＃ＨＥＰ２２０−ＭＷ２４）をＩｎｃｕｂａｔｉｏｎ Ｍｅｄｉｕｍ（製品名：Ｂｉｏｐｒｅｄｉｃ Ｉｎｔｅｒｎａｔｉｏｎａｌ， ＃ＭＩＬ２１４）で３日培養したのち、ｃ−Ｍｅｔ阻害剤ＰＦ−０４２１７９０３（終濃度１００ ｎｍｏｌ／Ｌ）（Ｓｅｌｌｅｃｋ Ｃｈｅｍｉｃａｌｓ， ＃Ｓ１０９４， ｌｏｔ． ０５）または媒体（ビヒクル：ＤＭＳＯ）を添加し、１時間後にｒｈ−ＨＧＦ（終濃度１００， ３００， １０００ ｎｇ／ｍＬ）またはコントロール群（Ｃｏｎｔｒｏｌ）には、（Ｉｎｃｕｂａｔｉｏｎ Ｍｅｄｉｕｍ）を添加した。 Experiment:
Human primary hepatocytes seeded in treated 24-well plates with human primary hepatocytes and rh-HGF (3.8 × 10 5 cells / well, Biopredic International, # HEP220-MW24 ) a Incubation Medium (product name: Biopredic International , # MIL214) for 3 days, then the c-Met inhibitor PF-04217903 (final concentration 100 nmol / L) (Selleck Chemicals, # S1094, lot. 05) or medium (vehicle: DMSO) was added and 1 After hours, (Incubation Medium) was added to the rh-HGF (final concentration 100, 300, 1000 ng / mL) or control group (Control).
ヒトＡｐｏＡ４ ｍＲＮＡの定量
ｒｈ−ＨＧＦ添加２４時間後の肝細胞からＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ（ＱＩＡＧＥＮ， ＃７４１０４）およびＲＮａｓｅ−Ｆｒｅｅ ＤＮａｓｅ ｓｅｔ（ＱＩＡＧＥＮ， ＃７９２５４）を用いてｔｏｔａｌ ＲＮＡを精製し、ＲＮＡ濃度を吸光度により測定した。精製したｔｏｔａｌ ＲＮＡをＨｉｇｈ−Ｃａｐａｃｉｔｙ ｃＤＮＡ Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔｉｏｎ Ｋｉｔ ｗｉｔｈ ＲＮａｓｅ Ｉｎｈｉｂｉｔｏｒ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ， ＃４３７４９６６）を使用して逆転写しｃＤＮＡを作製した。得られたｃＤＮＡを鋳型として、ＡｐｏＡ４ ｍＲＮＡ発現量をリアルタイムＰＣＲ法で検討した。検討にあたっては、１８Ｓ ｒＲＮＡを内部コントロールとした。ＰＣＲ反応は、ＴａｑＭａｎ Ｆａｓｔ Ａｄｖａｎｃｅｄ Ｍａｓｔｅｒ Ｍｉｘ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ， ＃４４４４５５７）とＴａｑＭａｎ Ａｓｓａｙ（ＡｐｏＡ４： Ｈｓ００１６６６３６＿ｍ１， １８Ｓ ｒＲＮＡ： Ｈｓ９９９９９９０１＿ｓ１， Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いて、ＶｉｉＡ７リアルタイムＰＣＲシステム（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を使用して、ＰＣＲの増幅反応を実施した。増幅反応は、９５℃で２０秒インキュベートした後、９５℃で１秒、６０℃で２０秒の反応を４０サイクル繰り返した。ＡｐｏＡ４ ｍＲＮＡの発現値をサンプルごとに１８Ｓ ｒＲＮＡ発現値で標準化することで、サンプル間の補正を実施した。 Quantification of human ApoA4 mRNA Total RNA was purified from hepatocytes 24 hours after the addition of rh-HGF using RNeasy Mini Kit (QIAGEN, # 74104) and RNase-Free DNase set (QIAGEN, # 79254), and the RNA concentration was absorbed. Measured by. The purified total RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, # 4374966) to prepare cDNA. Using the obtained cDNA as a template, the expression level of ApoA4 mRNA was examined by a real-time PCR method. In the study, 18S rRNA was used as an internal control. PCR reaction is performed using TaqMan Fast Advanced Master Mix (Applied Biosystems, # 4444557) and TaqMan Assay (ApoA4: Hs00166663_m1, 18S rRNA: Hs999999601_s, Real-time System, Hs99999911_s1, Amplification reaction was carried out. In the amplification reaction, after incubating at 95 ° C. for 20 seconds, the reaction at 95 ° C. for 1 second and 60 ° C. for 20 seconds was repeated for 40 cycles. Corrections between samples were performed by standardizing the expression value of ApoA4 mRNA at the expression value of 18S rRNA for each sample.
統計解析：
ＡｐｏＡ４ ｍＲＮＡ定量の結果について、コントロールとｒｈ−ＨＧＦ投与群との間で、ダネットの多重比較検定を実施した。ＡｐｏＡ４ ｍＲＮＡが統計的に有意に増加したｒｈ−ＨＧＦ処置群について、ビヒクル処置群とｃ−Ｍｅｔ阻害剤処置群との間で対応のないｔ検定を実施した（図５）。 Statistical analysis:
For the results of ApoA4 mRNA quantification, a multiple comparison test of Danette was performed between the control and the rh-HGF-administered group. An unpaired t-test was performed between the vehicle-treated group and the c-Met inhibitor-treated group for the rh-HGF-treated group in which ApoA4 mRNA was statistically significantly increased (Fig. 5).
結果：
ヒト初代培養肝細胞をｒｈ−ＨＧＦに暴露することによって、ヒトＡｐｏＡ４ ｍＲＮＡの発現が強力に増強された。また、その増強効果は、特異的ｃ−Ｍｅｔ阻害剤によって阻害されたことから、ＡｐｏＡ４はヒト肝細胞においてもｃ−Ｍｅｔ介在性にｒｈ−ＨＧＦ生物活性を反映したＰＤマーカーであることが強く示唆された。従って、ＡｐｏＡ４はヒトにおいてもｒｈ−ＨＧＦのＰＤマーカーとなることが示唆された（図５）。 result:
Exposure of human primary cultured hepatocytes to rh-HGF strongly enhanced the expression of human ApoA4 mRNA. In addition, since its enhancing effect was inhibited by a specific c-Met inhibitor, it is strongly suggested that ApoA4 is a PD marker that reflects rh-HGF bioactivity in c-Met-mediated manner even in human hepatocytes. Was done. Therefore, it was suggested that ApoA4 is also a PD marker for rh-HGF in humans (Fig. 5).
ヒトＡｐｏＡ４タンパク質の定量
ｒｈ−ＨＧＦ添加２４時間後の肝細胞培養上清（ｍＲＮＡ調製用の細胞と同一物の培養上清）をもちいて、ウェスタンブロット法によってＡｐｏＡ４タンパク質を定量した。ＰＢＳ（Ｗａｋｏ）で４倍希釈した培養上清に１／４容の泳動バッファー液（３１３ ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８），１０％ ＳＤＳ，３０％ Ｇｌｙｃｅｒｏｌ，０．２ ｍｇ／ｍＬ Ｂｒｏｍｏ−ｐｈｅｎｏｌ ｂｌｕｅ，２５％ ２−Ｍｅｒｃｕｐｔｏｅｔｈｎｏｌ）を添加し（培養上清の最終希釈度は２０倍となる）、９５℃、３分加熱処理した。その内 １０μＬをＳＤＳ−ＰＡＧＥに供し、ニトロセルロースフィルター（Ｓｃｈｌｅｉｃｈｅｒ＆Ｓｃｈｕｅｌｌ， ＢＡ８５）にブロッティングした。フィルターをブロッキング液（５０ ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５），１５０ ｍＭ ＮａＣｌ，５％ ｓｋｉｍ−ｍｉｌｋ，０．０１％ Ｔｗｅｅｎ２０，０．１％ ＮａＮ３）に浸し、室温で１時間振とうしてからブロッキング液を廃棄し、ｒａｂｂｉｔ ａｎｔｉ−ｈｕｍａｎ Ａｐｏｌｉｐｏｐｒｏｔｅｉｎ Ａ４抗体（Ｐｒｏｔｅｉｎｔｅｃｈ， １７９９６−１−ＡＰ）を０．０６７ μｇ／ｍＬ濃度で添加した反応溶液（５０ ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５），１５０ ｍＭ ＮａＣｌ，５％ ｓｋｉｍ−ｍｉｌｋ，４％ ＰＥＧ６０００，０．２％ ＥＤＴＡ／３Ｎａ，０．０１％ Ｔｗｅｅｎ２０，０．２％ Ｐｒｏｃｌｉｎ１５０）に浸し、室温下で２時間振とうした。洗浄液（５０ ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５），１５０ ｍＭ ＮａＣｌ，０．０１％ Ｔｗｅｅｎ２０）で３回洗浄した後、二次抗体のＨＲＰ標識ｇｏａｔ ａｎｔｉ−ｒａｂｂｉｔ ＩｇＧ（Ｈ＋Ｌ）（Ｊａｃｋｓｏｎ）を１／２０００濃度で添加した上記反応溶液に浸し、室温で１時間振とうした。上記洗浄液で３回洗浄した後、化学発光溶液ＳｕｐｅｒＳｉｇｎａｌ Ｗｅｓｔ Ｄｕｒａ Ｅｘｔｅｎｄｅｄ Ｄｕｒａｔｉｏｎ Ｓｕｂｓｔｒａｔｅ（Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ）を加えて発光させ、ＬＡＳ−４０００（ＦＵＪＩＦＩＬＭ）でＡｐｏＡ４タンパク質を検出した。 Quantification of human ApoA4 protein ApoA4 protein was quantified by Western blotting using hepatocyte culture supernatant (culture supernatant of the same cell as cells for mRNA preparation) 24 hours after addition of rh-HGF. 1/4 volume of migration buffer (313 mM Tris-HCl (pH 6.8), 10% SDS, 30% Glycerol, 0.2 mg / mL Bromo-phenol) in culture supernatant diluted 4-fold with PBS (Wako) Blue, 25% 2-Mercuptoethnor) was added (the final dilution of the culture supernatant was 20-fold), and the cells were heat-treated at 95 ° C. for 3 minutes. Of this, 10 μL was subjected to SDS-PAGE and blotting was performed on a nitrocellulose filter (Schleicher & Schuell, BA85). Immerse the filter in blocking solution (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% skim-milk, 0.01% Tween 20, 0.1% NaN3) and shake at room temperature for 1 hour. Reaction solution (50 mM Tris-HCl (pH 7.5), 150 mM NaCl) in which the blocking solution was discarded and rabbit anti-human Apolipoprotein A4 antibody (Proteintech, 17996-1-AP) was added at a concentration of 0.067 μg / mL. , 5% skim-milk, 4% PEG6000, 0.2% EDTA / 3Na, 0.01% Tween20, 0.2% Proclin150) and shaken at room temperature for 2 hours. After washing 3 times with a washing solution (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.01% Room 20), HRP-labeled goat anti-rabbit IgG (H + L) (Jackson) of the secondary antibody was added to 1 /. It was immersed in the above reaction solution added at a concentration of 2000 and shaken at room temperature for 1 hour. After washing with the above washing solution three times, the chemiluminescent solution SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) was added to cause light emission, and the ApoA4 protein was detected by LAS-4000 (FUJIFILM).
統計解析：
ＡｐｏＡ４タンパク質の定量結果について、コントロールとｒｈ-ＨＧＦ投与群との間で、ダネットの多重比較検定を実施した。で、ＡｐｏＡ４タンパク質が統計的に有意に増加したｒｈ−ＨＧＦ処置群について、ビヒクル処置群とｃ−Ｍｅｔ阻害剤処置群との間で対応のないｔ検定を実施した（図６）。 Statistical analysis:
For the quantification results of ApoA4 protein, a multiple comparison test of Danette was performed between the control and the rh-HGF-administered group. In the rh-HGF-treated group in which the ApoA4 protein was statistically significantly increased, an unpaired t-test was performed between the vehicle-treated group and the c-Met inhibitor-treated group (Fig. 6).
結果２：
ヒト初代培養肝細胞をｒｈ−ＨＧＦに暴露することによって、ヒトＡｐｏＡ４タンパク質の細胞外への放出が強力に増強された。また、その増強効果は、特異的ｃ−Ｍｅｔ阻害剤によって阻害されたことから、ＡｐｏＡ４タンパク質はヒト肝細胞においてもｃ−Ｍｅｔ介在性にｒｈ−ＨＧＦ生物活性を反映したＰＤマーカーであることが強く示唆された。従って、ＡｐｏＡ４タンパク質はヒトにおいてもｒｈ−ＨＧＦのＰＤマーカーとなることが示唆された（図６）。
Result 2:
Exposure of human primary cultured hepatocytes to rh-HGF strongly enhanced the extracellular release of human ApoA4 protein. In addition, since its enhancing effect was inhibited by a specific c-Met inhibitor, the ApoA4 protein is strongly a PD marker that reflects rh-HGF bioactivity in a c-Met-mediated manner even in human hepatocytes. It was suggested. Therefore, it was suggested that the ApoA4 protein is also a PD marker for rh-HGF in humans (Fig. 6).
Claims (9)
ＨＧＦが投与された被験体から採取した生体試料中のＡｐｏＡ４タンパク質の発現レベルを測定するステップ、
測定した前記生体試料中のＡｐｏＡ４タンパク質の発現レベルを、ＨＧＦが投与されていない、または、その投与の影響が消失した被験体から採取した前記生体試料中のＡｐｏＡ４タンパク質の発現レベルと比較するステップ、およびＨＧＦが投与されていない、または、その投与の影響が消失した被験体のＡｐｏＡ４タンパク質の発現レベルと比較して、ＨＧＦが投与された被験体のＡｐｏＡ４タンパク質の発現レベルが上昇している場合に、当該投与されたＨＧＦが薬力学的作用をもたらす量で投与されていると判定するステップ
を含み、
ここで、前記生体試料は、血液由来の生体試料である、
ことを特徴とする、
評価方法。 A method for evaluating the pharmacodynamic effects of hepatocyte growth factor (HGF).
A step of measuring the expression level of ApoA4 protein in a biological sample collected from a subject to which HGF was administered.
A step of comparing the measured expression level of ApoA4 protein in the biological sample with the expression level of ApoA4 protein in the biological sample collected from a subject who has not been administered HGF or whose effect of administration has disappeared. And when the expression level of ApoA4 protein in the subject to whom HGF was administered is elevated compared to the expression level of ApoA4 protein in the subject to whom HGF was not administered or the effect of the administration disappeared. , Including the step of determining that the administered HGF is administered in an amount that produces a medicinal effect.
Here, the biological sample is a biological sample derived from blood.
Characterized by
Evaluation method.
ＨＧＦが投与された被験体から採取した生体試料中のＡｐｏＡ４遺伝子またはＡｐｏＡ４タンパク質の発現レベルを測定するステップ、
測定した前記生体試料中のＡｐｏＡ４遺伝子またはＡｐｏＡ４タンパク質の発現レベルを、ＨＧＦが投与されていない、または、その投与の影響が消失した被験体から採取した前記生体試料中のＡｐｏＡ４遺伝子またはＡｐｏＡ４タンパク質の発現レベルと比較するステップ、および
ＨＧＦが投与されていない、または、その投与の影響が消失した被験体のＡｐｏＡ４遺伝子またはＡｐｏＡ４タンパク質の発現レベルと比較して、ＨＧＦが投与された被験体のＡｐｏＡ４遺伝子またはＡｐｏＡ４タンパク質の発現レベルが上昇している場合に、当該投与されたＨＧＦが薬力学的作用をもたらす量で投与されていると判定するステップ
を含み、
ここで、前記生体試料は、肝臓由来の生体試料である、
ことを特徴とする、
評価方法。 A method for evaluating the pharmacodynamic effects of hepatocyte growth factor (HGF).
A step of measuring the expression level of ApoA4 gene or ApoA4 protein in a biological sample collected from a subject to which HGF has been administered.
The measured expression level of the ApoA4 gene or ApoA4 protein in the biological sample is the expression of the ApoA4 gene or ApoA4 protein in the biological sample collected from a subject to whom HGF has not been administered or the effect of the administration has disappeared. Steps to compare with levels, and the expression level of the ApoA4 gene or ApoA4 protein of a subject who has not been administered HGF or whose effects have disappeared, or the ApoA4 gene of a subject who has been administered HGF. Including the step of determining that the administered HGF is administered in an amount that produces a medicinal effect when the expression level of the ApoA4 protein is elevated.
Here, the biological sample is a biological sample derived from the liver.
Characterized by
Evaluation method.
前記生体試料中のＡｐｏＡ４タンパク質の発現レベルが、イムノアッセイによって測定される、
ことを特徴とする、
評価方法。 The evaluation method according to claim 1 or 2.
The expression level of ApoA4 protein in the biological sample is measured by an immunoassay.
Characterized by
Evaluation method.
前記生体試料中のＡｐｏＡ４タンパク質の発現レベルが、イムノアッセイによって測定され、
前記生体試料は、血清または血漿に由来する試料である、
ことを特徴とする、
評価方法。 The evaluation method according to claim 1 .
The expression level of ApoA4 protein in the biological sample was measured by an immunoassay.
The biological sample is a sample derived from serum or plasma.
Characterized by
Evaluation method.
前記生体試料中のＡｐｏＡ４遺伝子の発現レベルが、リアルタイムＰＣＲによって測定される、
ことを特徴とする、
評価方法。 The evaluation method according to claim 2.
The expression level of the ApoA4 gene in the biological sample is measured by real-time PCR.
Characterized by
Evaluation method.
前記生体試料は、肝細胞または肝組織である、
ことを特徴とする、
評価方法。 The evaluation method according to claim 2 or 5.
The biological sample is hepatocytes or liver tissue.
Characterized by
Evaluation method.
前記被験体は、健常人である、
ことを特徴とする、
評価方法。 The evaluation method according to any one of claims 1 to 6.
The subject is a healthy person,
Characterized by
Evaluation method.
前記被験体は、肝臓疾患を有する被験体である
ことを特徴とする、
評価方法。 The evaluation method according to any one of claims 1 to 7.
The subject is characterized by having a liver disease.
Evaluation method.
抗ＡｐｏＡ４抗体を含む、
ことを特徴とする、
キット。 A kit for evaluating the pharmacodynamic effects of HGF
Contains anti-ApoA4 antibody,
Characterized by
kit.
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| US9272018B2 (en) * | 2011-04-18 | 2016-03-01 | Kyoto University | Acute hepatic insufficiency depressant and method for evaluating drug efficacy thereof |
| WO2013001885A1 (en) * | 2011-06-27 | 2013-01-03 | コニカミノルタホールディングス株式会社 | Image processing device, image processing method, and program |
| WO2013018851A1 (en) * | 2011-08-02 | 2013-02-07 | 独立行政法人国立がん研究センター | Method for inducing hepatic differentiation from induced hepatic stem cell, and induced hepatic progenitor cell |
-
2017
- 2017-03-16 US US16/078,557 patent/US20200341014A1/en not_active Abandoned
- 2017-03-16 WO PCT/JP2017/010587 patent/WO2017159771A1/en active Application Filing
- 2017-03-16 EP EP17766767.2A patent/EP3431992A4/en not_active Withdrawn
- 2017-03-16 JP JP2018505994A patent/JP6800208B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| WO2017159771A1 (en) | 2017-09-21 |
| JPWO2017159771A1 (en) | 2019-01-31 |
| US20200341014A1 (en) | 2020-10-29 |
| EP3431992A4 (en) | 2019-10-23 |
| EP3431992A1 (en) | 2019-01-23 |
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